Antigenic modification of polypeptides

ABSTRACT

Modified hormones or fragments of hormones are useful in producing antibodies when administered to an animal. Said antibodies in turn cause neutralization of endogenous natural protein hormones. The modification may be accomplished by attaching various kinds of modifying groups to the hormone or fragment. Modification may, for example, be achieved by chemically coupling diazosulfanilic acid groups to the hormone or fragment. The protein hormones to which this procedure can be applied are mammalian protein reproductive hormones such as, for example, Follicle Stimulating Hormone (FSH), or Human Chorionic Gonadotropin (HGG). These modified hormone or fragment may be administered to animals for the purpose of contraception, abortion, or treatment of hormone related disease states and disorders.

This application is a continuation-in-part of Ser. No. 622,031 filedOct. 14, 1975, now abandoned which in turn is a continuation-in-part ofSer. No. 462,955 filed Apr. 22, 1974, which in turn is acontinuation-in-part of Ser. No. 406,821, filed Oct. 16, 1973, which inturn is a continuation-in-part of Ser. No. 357,892, filed May 7, 1973now abandoned.

BACKGROUND OF THE INVENTION

It is well known that antibodies are generated in humans and in otheranimals in response to the presence of foreign antigens. It is alsoknown to confer immunity on an animal by administering an antibodyformed elsewhere. For instance, the patents to Michaelson (U.S. No.3,553,317), Friedheim (U.S. No. 2,388,260), Reusser (U.S. No. 3,317,400)and Peterson (U.S. No. 3,376,198) relate to production of antibodies,which when injected into an animal of a different species or into ahuman being cause passive immunization. In patents to Fell (U.S. No.2,301,532 and U.S. No. 2,372,066), the patentee refers to activeimmunization using modified histamine in such animals as horses, cows,etc. In a paper by R. G. Edwards in the British Medical Journal, Vol.26, pages 72 to 78, published in 1970 on "Immunology of Conception andPregnancy", he surveys the literature regarding the possibilities ofutilizing immunological methods to influence or control fertility,surveying first production of antibodies against tests or spermatozoa.Much of the literature surveyed is directed to the production of foreignantibodies which are injected into the subject (passive immunization).

Hormone antibodies have been studied for a long time and the effect ofspecific antisera have been recorded for many years. It is known thatadministration of certain antibodies during pregnancy can suppressimplantation or cause fetal resorption. Several different approacheshave been tried ranging from the induction of near permanent infertilityin the case of agglutination of spermatozoa in the male to thedisturbance of a single pregnancy by passive immunization withantibodies.

There are serious limitations to the use of passive immunizationprocedures for human therapy. Since the antibodies are practicallyproduced only in non-human animals, the repeated injection of animalproteins into humans is known to produce serious reaction in manyindividuals.

British Patent Specification No. 1,058,828 discloses that smallmolecules, referred to as "serological determinant peptides", can becoupled to large protein molecules, such as cattle albumin and theresultant conjugate then may be injected into animals for antibodyproduction. The document lists proteins from which the serologicallydeterminant peptides may be isolated prior to being used in the processtaught, the collection including viruses and bacteria whose surfacecomponent has the characteristics of a protein, toxins and hormoneshaving protein structure and enzymes. No specific hormone is named inthe document and no utility of anti-hormone immunization is described.The patent specification references a publication entitled: "TheSpecificity of Serological Reactions", Dover Publications, Inc., NewYork, 1962, Chapter V, "Artificial Conjugated Antigens" by K.Landsteiner. This publication outlines various chemical methods andapplies them passively to bind various toxic substances in the bloodsuch as arsenic. Thyroxine data provided in the publication suggeststhat such methods may be applied to protein hormones without indicatingthe therapeutic application, the publication teaching that specificantibodies may be formed to the small molecules and these antibodies arecapable of neutralizing the biological action of a large protein fromwhich the small peptide was a part.

Recently it has been discovered that doses of certain steroidsconsisting of synthetic non-protein hormones ("The Pill") whenadministered at stated intervals usually confer protection againstpregnancy for a short time (possibly a month). This medication hassometimes been found to create undesirable side effects in creatingundesirable metabolic changes and sometimes changes in the bloodclotting mechanisms. Moreover, the effect of each dose is of such shortduration that often it is of limited application, particularly in remoteareas to persons not readily instructed on proper and continuing use.

There is need therefore of an effective safe method of creating atemporary but relatively long-time immunity against pregnancy which doesnot have serious side effects. There is also a need for an effectivesafe method of terminating a pregnancy soon after conception which doesnot have serious harmful side effects. Such need may be met by theneutralization of a reproductive protein which is necessary for thenormal events of conception and/or gestation.

There is also a need for a means for control of various disease statesor maladies caused or influenced by unusual excesses of certainpolypeptides such as gastrin, angiotension II, or somatomedian. It isbelieved that this invention meets this need safely and effectively.

SUMMARY OF THE INVENTION

This invention is concerned (1) with the production of antigens for thepurpose of active immunization, (2) with the antigens so produced, and(3) with the use of said antigens. More particularly, the inventionrelates to antigens consisting of natural protein reproductive hormones,non-hormonal proteins, specific fragments of such hormones and proteinsand synthetically derived portions of said hormones and proteins, allmodified as will be indicated more fully hereinafter. For the sake ofsimplicity, hereinafter in this specification and in the claims, theseantigens are collectively referred to as modified polypeptides.

The invention is directed in one aspect to the use of modifiedpolypeptides in actively immunizing an animal, particularly mammals,against the biological action of endogenous unmodified non-hormonalnatural protein and/or hormone. The state of immunity arises because ofthe creation of antibodies which act against both the antigenic modifiedpolypeptide and its endogenous counterpart which is neutralized(rendered biologically ineffectual) as a result of the existence of saidantibodies. The immunity may take place because of the inability of theantibody to distinguish between the modified polypeptide and thenaturally existing protein, but it is uncertain that this is in fact thesituation. In effect, the invention provides, in one aspect, for theisoimmunization of a primate animal.

A more specific aspect of this invention relates to the modification ofprotein reproductive hormones by adding certain numbers of foreignmoieties to each hormone molecule, or hormonal fragment. Themodification must be sufficient to cause the body to create antibodiesto the modified hormones which will neutralize or inhibit the biologicalaction of the natural hormones produced by the body. Thus, the modifiedhormones become antigenic and cause the production of antibodies whichdisrupt the natural processes of conception and/or gestation. The term"protein reproductive hormones" includes those hormones essential to thenormal events of the reproductive process.

According to a further aspect of this invention, a disease state whichcan be treated by application of the technique of the instant inventionis the digestive disorder known to those skilled in the medical field asthe Zollinger-Ellison Syndrome. This syndrome or disease state isgenerally described as a condition in which a hyper secretion of thepolypeptide gastrin, which is produced in the pancreas and brings abouta state of hyperacidity in the stomach which results in a chronicdigestive disorder. Heretofore, the only effective treatment for thisdisease state was the surgical removal of a part or total removal of thesubject's stomach. Although survival of such patients is usually notthreatened, the medical state and life style of such individuals isseverely affected by such treatment.

Treatment of such subjects with hapten coupled (produced according tothe general method described herein) or otherwise chemically modifiedgastrin can be used to enhance the production of antibodies against thehypersecretion of gastrin and thereby alleviate or reduce the symptomsof this disease without surgical intervention. Sufficient reduction byimmunological means of this substance in the system of the body would besufficient to avoid the complicated and serious consequences of thesurgical treatment currently in use. In practice, an effective amount ofmodified gastrin is simply injected into the patient as required toaccomplish the control of the flow or presence of gastrin.

Another serious medical problem which is treatable by the application ofthe technique of the instant invention is that of hypertension. Ingeneral terms, the state of hypertension is the abnormal level orfluctuation of one's blood pressure. The blood pressure in an individualis controlled by many physiological processes in the body. However, onemajor substance affecting the regulation of such pressure is thehormonal polypeptide known as angiotension II. In certain states of highblood pressure (hypertension) it is difficult to medically control thesecretion and therefore the level of angiotension II in the circulatorysystem. By the appropriate modification of this hormone and subsequentimmunization with this altered modified proteinaceous hormone, it ispossible to reduce the secretion of angiotension II in patients withchronically elevated hormone levels. The predictable and controlledreduction of this substance is beneficial to certain patients withchronic problems of hypertension. Modified angiotension II can beproduced by the general protein modification technique described herein.The resultant modified angiotension II is simply injected into thepatient in an amount sufficient to induce antibody response sufficientto control or regulate unmodified angiotension II to the desired degree.

A further embodiment of the present invention is the treatment ofdiabetes and associated micro and macro vascular diseases. Currently,the treatment of diabetes is limited to dietary and/or drug treatment toregulate blood glucose levels. Recent scientific data support theconcept that growth hormone and somatomedian (both polypeptides) areintimately involved in the disease syndrome. These substances can bemodified by the technique described herein and used in an effectiveamount to control the progress of this disease. In practice, modifiedgrowth hormone or modified somatomedian is injected into the body todevelop antibodies for control of the normally secreted hormones.

Another health problem that can be treated by the use of the concepts ofthis invention is that of certain endocrine or hormone dependent breasttumors or cancers. Certain of these cancers have been shown to bedependent upon the abundant secretion of the hormone prolactin for theircontinued survival. The inhibition of the secretion of prolactin hasbeen shown to diminish the growth rate and the actual survival ofcertain of these tumors. The immunization of such subjects with thehapten coupled or otherwise altered prolactin produced as describedherein, would result in the systematic reduction of the level of thishormone circulating in the system and consequently, may result in theregression or remission of tumor growth. The consequence of thistreatment would be far more favorable in terms of effective treatment ofthis disease since surgical removal of the breasts is a principal methodof treatment currently available. It should be understood that thistreatment should be effective for only those tumors that are dependentupon the secretion of prolactin for survival.

Investigators also have determined, for example, that certainpolypeptide entities are supportive factors to and secretions ofneoplastic diseases in both man and other animals. These entities havebiochemically, biologically and immunologically close resemblances tohormones, particularly to Chorionic Gonadotropin (CG), as well as toLuteinizing Hormone (LH). By applying the isoimmunization techniques ofthe invention, the function of such polypeptides or endogenouscounterparts can be neutralized to carry out regulation of themalignancy. For example, tumors in both male and female primates may betreated by isoimmunization procedures developing antibodies to ChorionicGonadotropin or Luteinizing Hormone or the noted entity analogousthereto. Further, neoplasms in primate females may be regulated byisoimmunization procedures developing antibodies to endogenous FollicleStimulating Hormone (FSH). This hormone, when associated with a tumorstate, tends to aggravate the tumorous condition.

The immunochemical control asserted, as noted, neutralizes the naturallyoccuring hormone or the above-described entity biologically analogousthereto. As a consequence, the hormone or entity will not be availableas would normally be the case, for example, the stimulation of someaction of a target tissue. Conversely, the neutralization of thebiological activity of the hormone or analogous entity may serve to takeaway an inhibitory action which it otherwise might assert.

There are certain other disease states that may be treatable by the useof altered or modified hormonal or non-hormonal proteins as antigens.The disease states and the associated substances that may be used asmodified antigens for immunological treatment of these diseases will belisted as follows:

(1) modified parathyroid hormone for the treatment of kidney stones,

(2) modified insulin and/or glucagon for the treatment ofhyperinsulinoma,

(3) modified thyroid stimulating hormone (TSH) for the treatment ofhyperthyroidism, and

(4) modified secretin for the treatment of irritable bowel syndrome.

Another group of polypeptides which can be altered by the proceduresdescribed herein and used in the field of human fertility control arespecific non-hormonal protein antigens isolated from placental tissue.There is direct evidence that inhibition of substances that are specificto the placental tissue and do not have similar antigenic propertieswith other antigens from organs in other parts of the body, can resultin the disruption of pregnancies by passive immunization. Such specificplacental substances when modified to form modified polypeptides by theprocedures described herein can be injected into the body of an animalof the same species as an effective fertility control means with themechanism being active immunization similar to that described for theantigenic modification of hormones. The particular advantage of thesesubstances is that placental antigens are foreign to the non-pregnantfemale human subject and therefore are unlikely to cause anycross-reaction or disruption of normal body function in the non-pregnantfemale.

While the invention is useful for the human species it will beappreciated that it is also useful in connection with other animals.Similarly, while the reference herein with respect to fertility controlis primarily directed to females, such described techniques may beapplicable to males, i.e. FSH, its beta subunit and fragments thereof.Such immunization represents an effective fertility control procedure,providing no physiological consequences are encountered which may befound to react adversely to the performance of other body constituents.

Whether the concerned hormone, non-hormonal protein or specific fragmentthereof which is modified is naturally occurring or is a syntheticproduct is clearly immaterial. A synthetic protein molecule will performthe same function as the naturally occurring one, inasmuch as the bodywill react in an equivalent antigenic manner.

It has accordingly been discovered by virtue of this invention that itis possible to interfere with or treat various disease states or medicalproblems which are caused or influenced by certain polypeptides byactive immunization of a male or female animal by the reproduction anduse of antigens formed by administration of modified polypeptides. Themodification of the polypeptides forms antigens which are thanadministered into an animal in which immunization is to be developed.Said modification is accomplished by attaching to a polypeptide one ormore foreign reactive (modifying) groups and/or by attaching two or morepolypeptides to a foreign reactive group (i.e., a carrier) or both ofthe above, so that the body of the animal, recognizing the modifiedpolypeptide as a foreign object, produces antibodies which neutralizenot only the modified protein but also the natural protein which isresponsible for the disease or medical problem being regulated. In orderto produce an effective quanta of antibodies to the antigen or targetedfunctional polypeptide, it may be advantageous to administer themodified polypeptide together with an immunological adjuvant. The term"adjuvant" is commonly referred to by those engaged in the field at handas being a substance which will elevate the total immune response of ananimal or person to any immunization thereof, i.e. the adjuvant is anonspecific immuno-stimulator.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a chart describing the results of mating four baboons threetimes following the administration thereto of a fertility controllingantigen according to the invention;

FIG. 2 shows two plots illustrating the antifertility antibody levelsmaintained within two baboons following the administration of antigensthereto formulated in accordance with the invention; and

FIG. 3 shows three dose response lines illustrating the specificity ofantibody response to a CG antigen formulated in accordance with theinvention.

GENERAL DESCRIPTION

In an effort to better define the modified polypeptides with which thisinvention is concerned, it is first considered appropriate to set outmore precisely than hereinabove, examples of the natural hormones andnatural non-hormonal proteins modified according to this invention. Theyinclude Follicle Stimulating Hormone (FSH), Luteinizing Hormone (LH),Chorionic Ganadotropin (CG), e.g. Human Chorionic Ganadotropin (HCG),Placental Lactogen, e.g. Human Placental Lactogen (HPL) Prolactin, e.g.Human Prolactin, (all of which are proteinaceous reproductive hormones),gastrin, angiotension II, growth hormone, somatomedian, parathyroidhormone, insulin, glucagon, thyroid stimulating hormone (TSH), secretin,and other polypeptides which could adversely affect body function.

The hormone, Chorionic Gonadotropin (CG) has been the subject ofextensive investigation, it being demonstrated in 1927 that the bloodand urine of pregnant women contained a gonad-stimulating substancewhich, when injected into laboratory animals, produced marked gonadalgrowth. Later, investigators demonstrated with certainty that thePlacental Chorionic villi, as opposed to the pituitary, were the sourceof this hormone. Thus, the name Chorionic Gonadotropin or, in the caseof humans, Human Chorionic Gonadotropin (HCG) was given to this hormoneof pregnancy. During the more recent past, a broadened variety ofstudies have been conducted to describe levels of HCG in normal andabnormal physiological states, indicating its role in maintainingpregnancy. The studies have shown the hormones' ability to induceovulation and to stimulate corpus luteum function and evidence has beenevoked for showing its ability to suppress lymphocyte action. Theimmunological properties of the HCG molecule also have been studiedwidely. Cross-reaction of antibodies to HCG with human pituitaryLuteinizing Hormone (LH), and vice-versa, have been extensivelydocumented, see for example:

Paul , W. E. & Ross, F. T. (1964) Immunologic Cross Reaction Between HCGand Human Pituitary Gonadotropin. Endrocrinology, Vol. 75, pp. 352-358.

Flux, D. X. & Li C. H. (1965) Immunological Cross Reaction AmongGonadotropins. Acta Endrocrinologica, Vol. 48, pp. 61-72.

Bogshawe, K. D.; Orr, A. H. & Godden J. (1968) Cross-Reaction inRadi-Immunoassay between HCG and Plasma from Various Species. Journal ofEndocrinology, Vol. 42, pp. 513-518.

Franchimont, P. (1970) Study on the Cross-Reaction between HCG andPituitary LH. European Journal of Clinical Investigation, Vol. 1, pp.65-68.

Dorner, M.; Brossmer, R.; Hilgenfeldt, U. & Trude, E. (1972).Immunological reactions of Antibodies to HCG with HCG and its chemicalderivatives. In Structure-Activity Relationships of Proteins andPolypeptide Hormones (ed. M. Margoulies & F. C. Greenwood), pp. 539, 541Amsterdam: Exerpta Medica Foundation.

Further, these cross-reactions have been used to perform immunoassaysfor both CG and LM hormones. See:

Midgley, A. R. Jr. (1966) Radioimmunoassay: a method for HCG and LH.Endocrinology, Vol. 79, pp. 10-16.

Crosignani, P. G., Polvani, F. & Saracci R. (1969) Characteristics of aradioimmunoassay for HCE-LH. In Protein and Polypeptide Hormones (ed. M.Margoulies) pp 409, 411 Amsterdam: Excerpta Medica Foundation.

Isojima, S; Nake, O.; Kojama, K. & Adachi, H. (1970). Rapidradioimmunoassay of human L.H. using polymerized anti-human HCG asimmunoadsorbent. Journal of Clinical Endocrinology and Metabolism, Vol.31, pp. 693-699.

In addition to providing for the modification of the entire hormone orselected polypeptide, the invention further provides for the utilizationof modified subunits, for example the beta subunit of ChorinonicGonadotropin. Of particular interest, such subunits may be fragmentedinto smaller components herein termed "fragments". The latter can beproduced synthetically to exhibit an amino acid sequence sufficiently inanalogous correspondence to a predetermined portion of the parentsubunit. Such fragments generally are conjugated with a larger moleculeor component foreign to the body, which may be termed a "carrier", inorder to effectively evoke or raise a sufficient quanta of antibodies.The use of the fragments, as thus conjugated, advantageously provides ahigh degree of specificity of antigenic reaction to the targeted hormoneor its biochemical equivalent, i.e. the antibodies will not react withother body constituents. Of particular interest, the above-discussedcross reaction of HCG and LH can be avoided by utilization of fragmentsof the respective hormone due to the desirable specificity of responsethereto. Thus, when interested in obtaining an immunological reactionagainst the hormone, HCG, the undesirable immune reaction to thenaturally occuring body constituent, LH, may be eliminated. Syntheticequivalents of the fragments offer enhanced practicality both from thestandpoint of production costs and necessary maintenance of purity.

As is indicated in the above discussion, when considered in isolationwith respect to conception and pregnancy, CG only is present in femaleprimates when they are in a post conception state. However, as discussedabove and later herein, an entity at least analogous thereto (havingsimilar immunological properties to HCG) is seen to be present inconjunction with malignancies.

Subunits and fragments of the proteinaceous reproductive hormonesinclude the beta subunit of natural Follicle Stimulating Hormone, thebeta subunit of natural Human Chorionic Gonadotropin, fragmentsincluding, inter alia, a 20-30 or 30-39 amino acid peptide consisting ofthe C-terminal residues of natural Human Chorionic Gonadotropin betasubunit, as well as specific unique fragments of natural Human Prolactinand natural Human Placental Lactogen, which may bear little resemblanceto analogous portions of other protein hormones. Further with respect tothe type of novel chemical entities with which this invention isconcerned, one may note for instance the chemical configuration of thebeta subunit of HCG. That structure is as follows:

Ser-Lys-Glu-Pro-Leu-Arg-Pro-Arg-Cys-Arg¹⁰-Pro-Ile-Asn*-Ala-Thr-Leu-Ala-Val-Glu-Lys²⁰-Glu-Gly-Cys-Pro-Val-Cys-Ile-Thr-Val-Asn*-Thr-Thr-Ile-Cys-Ala-Gly-Try-Cys-Pro-Thr⁴⁰-Met-Thr-Arg-Val-Leu-Gln-Gly-Val-Leu-Pro⁵⁰-Ala-Leu-Pro-Gln-Val-Val-Cys-Asn-Try-Arg⁶⁰-Asp-Val-Arg-Phe-Glu-Ser-Ile-Arg-Leu-Pro⁷⁰-Gly-Cys-Pro-Arg-Gly-Val-Asn-Pro-Val-Val⁸⁰-Ser-Tyr-Ala-Val-Ala-Leu-Ser-Cys-Gln-Cys⁹⁰-Ala-Leu-Cys-Arg-Arg-Ser-Thr-Thr-Asp-Cys¹⁰⁰-Gly-Gly-Pro-Lys-Asp-His-Pro-Leu-Thr-Cys¹¹⁰-Asp-Asp-Pro-Arg-Phe-Gln-Asp-Ser-Ser-Ser¹²⁰-Ser*-Lys-Ala-Pro-Pro-Pro-Ser*-Leu-Pro-Ser¹³⁰-Pro-Ser*-Arg-Leu-Pro-Gly-Pro-Ser*-Asp-Thr¹⁴⁰ -Pro-Ile-Leu-Pro-GlnStructure (I)

For specificity of antibody action it is necessary that distinctivepeptides be isolated or prepared that contain molecular structurescompletely or substantially completely different from the otherhormones. The beta-subunit of HCG possesses a specific chain or chainsor amino acid moieties which differ either completely or essentiallyfrom the polypeptide chain of Human Luteinizing Hormone. These chains orfragments, when conjugated with a carrier, represent an additionalaspect of this invention. Accordingly, the polypeptide Structures (II)and (III) [C-terminal portion of structure I)]

Asp-Asp-Pro-Arg-Phe-Gln-Asp-Ser-Ser-Ser-Ser*-Lys-Ala-Pro-Pro-Pro-Ser*-Leu-Pro-Ser-Pro-Ser*-Arg-Leu-Pro-Gly-Pro-Ser*-Asp-Thr-Pro-Ile-Leu-Pro-GlnStructure (II)Gln-Asp-Ser-Ser-Ser-Ser*-Lys-Ala-Pro-Pro-Pro-Ser*-Leu-Pro-Ser-Pro-Ser*-Arg-Leu-Pro-Gly-Pro-Ser*-Asp-Thr-Pro-Ile-Leu-Pro-GlnStructure (III)

whether obtained by purely synthetic methods or by enzymatic degradationfrom the natural or parent polypeptide, [Carlson et al., J. BiologicalChemistry, 284 (19), p. 6810, (1973)] when modified according to thisinvention, similarly provide materials with antigenic propertiessufficient to provide the desired immunological response. It will beunderstood, for example, that addition of a polytyrosine chain or aprotein macromolecule (carrier) may assist in rendering Structure (II)antigenic so that the resulting administration of modified Structure(II) will provide the desired immunological action against natural HCG.

The beta subunit set forth at Structure (I) is seen to represent achemical sequence of 145 amino acid components. This structure has ahigh degree of structural homology with the corresponding subunit ofLuteinizing Hormone (LH) to the extent of the initial 110 amino acidcomponents. As indicated above, it may be found desirable, therefore, toevoke a high specificity to the Chorionic Gonadotropin hormone or ananalogous entity through the use of fragments analogous to theC-terminal, 111-145 amino acid sequence of the subunit. Structure (II)above may be observed to represent just that sequence. Structure (III)is slightly shorter, representing the 116-145 amino acid positionswithin the subunit sequence.

Further polypeptide chains useful in promoting antibody buildup againstnatural HCG include the following structures labeled Structures (IV)through (XIV). When modified according to this disclosure, such as bycoupling to Ficoll 70* or other modifier-carriers such as proteinmacromolecules described herein, these polypeptides provide immunogenicactivity with which this invention is concerned. All of thesepolypeptides are considered fragments of HCG by virtue of theirsubstantial resemblance to the chemical configuration of the naturalhormone and the immunological response provided by them when modified asindicated herein.

Cys-Pro-Pro-Pro-Pro-Pro-Pro-Ser-Asp-Thr-Pro-Ile-Leu-Pro-Gln Structure(IV) Asp-Asp-Pro-Arg-Phe-Gln-Asp-Ser-Pro-Pro-Pro-Pro-Pro-Pro-CysStructure (V)Phe-Gln-Asp-Ser-Ser-Ser-Ser-Lys-Ala-Pro-Pro-Pro-Ser-Leu-Pro-Ser-Pro-Ser-Arg-Leu-Pro-Gly-Pro-Ser-Asp-Thr-Pro-Ile-Leu-Pro-GlnStructure (VI)Asp-Asp-Pro-Arg-Phe-Gln-Asp-Ser-Ser-Ser-Ser-Lys-Als-Pro-Pro-Pro-Ser-Leu-Pro-SerStructure (VII)Asp-Asp-Pro-Arg-Phe-Gln-Asp-Ser-Pro-Pro-Pro-Cys-Pro-Pro-Pro-Ser-Asp-Thr-Pro-Ile-Leu-Pro-GlnStructure (VIII)Asp-Asp-Pro-Arg-Phe-Gln-Asp-Ser-Pro-Pro-Pro-Pro-Pro-Pro-Cys-Pro-Pro-Pro-Pro-Pro-Pro-Ser-Asp-Thr-Pro-Ile-Leu-Pro-GlnStructure (VIIIa)Asp-His-Pro-Leu-Thr-Aba-Asp-Asp-Pro-Arg-Phe-Gln-Asp-Ser-Ser-Ser-Ser-Lys-Als-Pro-Pro-Pro-Ser-Leu-Pro-Ser-Pro-Ser-Arg-Leu-Pro-Gly-Pro-Ser-Asp-Thr-Pro-Ile-Leu-Pro-Gln-Pro-Pro-Pro-Pro-Pro-Pro-CysStructure (IX)Asp-Asp-Pro-Arg-Phe-Gln-Asp-Ser-Ser-Ser-Ser-Lys-Ala-Pro-Pro-Pro-Ser-Leu-Pro-Ser-Pro-Ser-Arg-Leu-Pro-Gly-Pro-Ser-Asp-Thr-Pro-Ile-Leu-Pro-Gln-Pro-Pro-Pro-Pro-Pro-Pro-CysStructure (X)Asp-Asp-Pro-Arg-Phe-Gln-Asp-Ser-Ser-Ser-Ser-Lys-Ala-Pro-Pro-Pro-Ser-Leu-Pro-Ser-Pro-Ser-Arg-Leu-Pro-Gly-Pro-Ser-Asp-Thr-Pro-Ile-Leu-Pro-Gln-CysStructure (XI)

Structure (IV) will be recognized as incorporating a Cys component atthe amino or N terminal which is associated with a Proline spacersequence. These spacers serve to position the sequence which followsphysically distant from the carrier-modifier. The latter sequence may beobserved to represent the 138th to 145th amino acid component sequenceof the subunit Structure (I). Structure (V) on the other hand,represents an initial sequence corresponding with the 111th to 118thcomponents of the subunit structure (I) followed by a sequence of sixProline spacer components and a carboxyl terminal, present as Cysteine.The rationale in providing such a structure is to eliminate theprovision of sites which may remain antigenically neutral inperformance. Structures (IV) and (V) represent relatively shorter aminoacid sequences to the extent that each serves to develop one determinantsite. Consequently, as alluded to in more detail hereinafter, they areutilized in conjunction with a mixed immunization technique wherein anecessary two distinct determinants are provided by the simultaneousadministration of two such fragments, each conjugated to acorresponding, separate carrier macromolecule. Structure (VI) representsthe 115th through 145th component sequence of structure (I). Structure(VII) represents a portion of Structure (I), however, essentially, asequence of the 111th to 130th components thereof is formed.

Structure (VIII) incorporates two sequences, one which may be recognizedin Structure (V) and the other in Structure IV. These two sequences areseparated by two spacer sequences of Proline components and one isjoined with an intermediately disposed Cysteine component which serves aconjugation function as described later herein. With the arrangement,two distinct determinant sites are developed in physically spacedrelationship to avoid the development of an unwanted artificialdeterminant possibly otherwise evolved in the vicinity of their mutualcoupling. Structure (VIIIa) represents Structure (VIII) with additionalPro spacer residues to provide a widened spacing of determinant sites.

Structure (IX) mimics sequences from Structure (I) with the addition ofa Proline Spacer Sequence, A Cysteine Component at the C-terminal, andan Aba substituted for Cys at the 110 position. The Aba designation isintended herein to mean alpha-aminobutyric acid of Cysteine. Structure(X) will be recognized as a combination of Structure (II) with a sixresidue Proline spacer sequence and a Cysteine component at theC-terminal. Similarly, Structure (XI) combines Structure (II) with aCysteine component at the C-terminal without a Proline spacer sequence.

Thr-Cys-Asp-Asp-Pro-Arg-Phe-Gln-Asp-Ser-Ser-Ser-Ser-Lys-Ala-Pro-Pro-Pro-Ser-Leu-Pro-Ser-Pro-Ser-Arg-Leu-Pro-Gly-Pro-Ser-Asp-Thr-Pro-Ile-Leu-Pro-GlnStructure (XII)Asp-His-Pro-Leu-Thr-Aba-Asp-Asp-Pro-Arg-Phe-Gln-Asp-Ser-Ser-Ser-Ser-Lys-Ala-Pro-Pro-Pro-Ser-Leu-Pro-Ser-Pro-Ser-Arg-Leu-Pro-Gly-Pro-Ser-Asp-Thr-Pro-Ile-Leu-Pro-Gln-CysStructure (XIII)Cys-Pro-Pro-Pro-Pro-Pro-Pro-Asp-Asp-Pro-Arg-Phe-Gln-Asp-Ser-Ser-Ser-Ser-Lys-Ala-Pro-Pro-Pro-Ser-Leu-Pro-Ser-Pro-Ser-Arg-Leu-Pro-Gly-Pro-Ser-Asp-Thr-Pro-Ile-Leu-Pro-GlnStructure (XIV)

Structure (XII) will be recognized as having the sequence of Structure(II) with the addition of Thr-Cys components at its N terminal.Structure (XIII) is similar to structure (IX) but does not contain thespacer conjugate. Structure (XIV) will be recognized as being similar toStructure II with the addition of spacer components at the N terminaland a Cys component for conjugation purposes.

Particularly where the larger whole hormone or subunit type molecularstructures are concerned, the number of foreign reactive groups whichare to be attached to the polypeptide and the number of polypeptides tobe attached to a foreign reaction group depends on the specific problembeing treated. Basically, what is required is that the concernedpolypeptide be modified to a degree sufficient to cause it to beantigenic when injected in the body of the host. If too littlemodification is effected, the body may not recognize the modifiedpolypeptide as a foreign body and would not create antibodies againstit. If the number of foreign molecules added to the polypeptides is toogreat, the body will create antibodies against the intruder antigen, butthe antibodies will be specific to the injected antigen and will notneutralize the action of the concerned natural endogenous hormone ornon-hormonal protein, i.e. they will be specific to the modifier.

In general, again considering the larger molecule subunit or wholehormone, it has been found that about 1-40 modifying groups per moleculeof polypeptide will be useful in modifying the polypeptide adequately soas to obtain the desired immunological effect of this invention. As willbe appreciated by one skilled in the art, this ratio of modifying groupsper polypeptide will vary depending upon whether an entire hormone isutilized for modification or whether for instance a relatively smallsynthetic fragment of said hormone is to be modified. Generally for thelarger molecules, it is preferred that 2-40 modifying groups permolecule of polypeptide be used according to this invention. In theinstance where the polypeptide is the β-subunit of HCG it isparticularly preferred that about 5-30 and more preferably 10-26modifying groups per molecule of polypeptide be used. The importantconsideration with respect to each modified polypeptide is that thedegree of modification be adequate to induce generation by the body ofantibodies adequate to neutralize some of the natural hormone ornon-hormonal protein against which neutralization is desired, and thiswill vary with each polypeptide involved, and the degree of correctionor change desired for the body function involved.

Modification of the polypeptide is accomplished by attaching variouskinds of modifying groups to proteinaceous hormone, non-hormonalproteins, subunits or specific fragments thereof according to methodsknown in the art.

As is apparent, structures (II)-(XIV) are relatively smaller fragments,usually produced synthetically. To render them capable of elicitingantibody production, it becomes necessary to conjugate them with largercarrier-modifier molecules. Generally about 5-30 peptide fragments willbe coupled with one carrier molecule. The body will, in effect,recognize these foreign carriers as well as the sequences represented bythe fragments and form antibodies both to the carrier and to thesequences of the coupled fragments. Note that the carrier-modifiers areforeign to the body and thus antibodies to them will not be harmful toany normal body constituents. In the latter regard, it may be foundpreferable to utilize a carrier which, through the development ofantibodies specific to it, will be found beneficial to the recipient.

As indicated earlier herein, it also is preferred that the modificationconstitute two or more immunological determinants represented on thenative hormone as polypeptide structures to which it is desired to evokean antibody response. The effect is one of heterogenity of antibodydevelopment. Thus, several fragment structures have been described abovehaving at least two distinct amino acid sequences represented in the HCGbeta subunit [Structure I]. These sequences may be so spaced as toderive the determinants in mutual isolation, while the spaced sequencefragment is conjugated with a larger, macromolecular carrier.Alternately, the noted mixed immunization arrangement may be utilizedwherein a first fragment developing one determinant is conjugated with afirst carrier molecule and is administered in combination with a second,distinct fragment which is conjugated with a second carrier molecule,the latter of which may be the same as or different in structure fromthe first carrier. Thus, each macromolecular carrier must be conjugatedwith hormone fragments such that each fragment represents two or moreimmunological determinants. These two necessary determinants can beevolved by mixing, for example, separate conjugate structures, forexample Structures (IV) and (V) each of which, through formingantibodies separately to the distinct determinants, will provide apopulation of antibodies reacting with two separate determinants on thenatural endogenous hormone.

Inasmuch as the noted fragments are relatively small as compared, forinstance, to a whole hormone or subunit thereof, a criterion of size isimposed upon the selection of a carrier. The carrier size must beadequate for the body immune system to recognize its foreign nature andraise antibodies to it. Additionally, carrier selection preferably ispredicated upon the noted antibody heterogeneity requirement, i.e. thecarrier must itself evoke a heterogeneous immune response in addition tothe fragments. For example, improved response may be recognized wherethe carrier is varied in structure, e.g. incorporating branching chainsto enhance the recognition of both the carrier and the attachedpolypeptide as being of a foreign nature.

As one example of whole hormone modification, modified diazo groupsderived from sulfanilic acid may be attached to the subjectpolypeptides, see the Cinader et al and Phillips et al references citedsubsequently for instruction on how this "attachment" is accomplished,and to the extent necessary for an understanding of this invention, suchis incorporated herein by reference.

Additional modifying groups for modifying whole hormones or theirsubunits are those groups obtained by reaction of the polypeptides withdinitrophenol, trinitrophenol, and S-acetomercaptosuccinic anhydride,while, suited for utilization as a carrier-modifier in conjunction withfragments, are polytyrosine in either straight or branched chains,polyalanines in straight or branched chains, biodegradable polydextran,e.g. polymerized sugars such as sucrose copolymerized withepichlorohydrin, e.g. Ficoll 70 and Ficoll 400* or a polyglucose such asDextran T 70**, serum proteins such as homologous serum albumin,hemocyanin from Keyhole limpet, a marine gastropod mollusk, viruses suchas influenza virus (type A, B, or C) or poliomyelitis virus, live orkilled, Types 1, 2 and 3 of tetanus toxoid, diphtheria toxiod, choleraorganisms or somewhat less preferably, natural proteins such asthyroglobulin, and the like. Generally, synthetic modifiers arepreferred over the natural modifiers. However, carrier-modifiers foundparticularly suitable for conjugation with the above-discussed fragmentstructures are Flagellin, tetanus toxoid and an influenza subunit, forexample, the preparation of which is described by Bachmeyer, Schmidt andLiehi, "Preparation and Properties of a Novel Influenza SubunitVaccine", Post-Graduate Medical Journal (June, 1976) 52:360-367. Thisinfluenza subunit was developed as a vaccine which incorporatesessentially only the two viral proteins, Haemagglutinin andNeuraminidase. Containing substantially only these two essentialimmunogens, the subunit represents a preparation which does not containother protein and lipid antigens which may be found to cause undesiredside reactions. A secondary benefit may be realized through theutilization for example, of the influenza subunit, poliomyelitis virus,tetanus toxoid, diphtheria toxoid, cholera antigens or the like as amodifier-carrier, inasmuch as beneficial antibodies will be raised tothat modifier-carrier as well as to the hormonal fragment conjugatedthereto.

Flagellin is a protein described as forming the wall of the main spiralfilament of the flagellum. Bacterial flagella, in turn, have been knownas the active organelles of cell locomotion, individual flagella(flagellum) occurring in suspension as individual spirals which, upondrying, collapse into filaments which describe a sine wave with a wavelength of 2-3 microns and an amplitude of 0.25-0.60 microns. Generally,the flagellum consists of three morphologically distinct parts: a basalstructure that is closely associated with the cytoplasmic membrane andcell wall, a hook and the noted main spiral filament.

Purified flagellum is readily obtained by solubilization of flagellarfilaments below a pH value of about four, and subsequent removal of theinsoluble material by centrifugation or filtration. As a group ofrelated proteins, flagellins from different bacterial species have beenpredicted to have similar amino-acid compositions. However, the aminoacid composition of each flagellin species is unique. Essentially allflagellins are described as containing no or only a few residues ofcysteine, tryptophan, tyrosine, proline and histidine. Thus, whenconjugated with fragments in accordance with the invention, athiolactonization procedure or the like is carried out as describedlater herein.

The molecular weights of various flagellin have been calculated, in allcases the values thereof of the monomeric subunits falling in the rangeof 30,000 to 50,000. From an immunological standpoint, a flagellinmolecule is highly immunogenic. For a further and more detaileddiscourse describing bacterial flagella and flagellin, reference is madeto "Advances in Microbial Physiology" 6:219 1971, "Bacterial Flagella"by R. W. Smith and Henry Coffler, which publication is incorporatedherein by reference.

Tetanus toxoids have been the subject of study and production for manyyears. The toxoid generally is evolved from a formalinization of tetanustoxin, the latter being a protein synthesized by Clostridium tetani.Immunization currently is carried out utilizing soluble and absorbedtetanus toxoids and suggestions have been made concerning theutilization of fluid tetanus toxoid in complex with antitoxin.Publications describing the toxin and toxoid are numerous, referencebeing made to the following:

1. Immunochemistry of Tetanus Toxin, Bizzini, et al, Journal ofBiochemistry, Vol. 39, pp. 171-181 (1973).

2. Early and Enhanced Antitoxin Responses Elicited with Complexes ofTetanus Toxoid and Specific Mouse and Human Antibodies, Stoner et al,Journal of Infectious Diseases, Vol. 131, No. 3, pp. 230-238 (1975).

3. Differences in Primary and Secondary Immunizability of Inbred MiceStrains, Ipsen, Journal of Immunology, Vol. 83, pp. 448-457 (1959);

4. Antigenic Thresholds of Antitoxin Responses Elicited in IrradiatedMice with Complexes of Tetanus Toxin and Specific Antibody, Hess et al,Radiation Research, Vol. 25, pp. 655-667 (1965).

5. Early and Enhanced Germinal Center Formation and Antibody Responsesin Mice After Primary Stimulation with Antigenisologous AntibodyComplexes as Compared with Antigen Alone, Laissue et al, Journal ofImmunology, Vol. 107, pp. 822-825, (1971).

6. Distinctive Medullary and Germinal Center Proliferative Patterns inMouse Lymph Nodes after Regional Primary and Secondary Stimulation withTetanus Toxoid, Buerki et al, Journal of Immunology, Vol. 112, No. 6,pp. 1961-1970 (1974)

Modification by removal of moieties is also contemplated by thisinvention. Thus, for example, where certain of the natural proteins havecarbohydrate moieties, these carbohydrate moieties may be removedaccording to methods known in the art by, for instance, N-acetylneuriminidase or N-acetyl glucosidase, materials useful for removal ofspecific carbohydrate moieties.

These various means for modification are, as indicated above, known topersons skilled in the art. Certain of these means may be found in thefollowing list of literature references, whereas various others of themmay be found elsewhere in the literature by art-skilled persons:

(1) Klotz et al., Arch. of Biochem. and Biophys., 96, pp. 605-612,(1966).

(2) Khorana, Chem. Rev. S3: 145, (1953).

(3) Sela et al., Biochem. J., 85, p. 223, (1962).

(4) Eisen et al., J. Am. Chem. Soc. 75, 4583, (1953).

(5) Centeno et al., Fed. Proc. (ABSTR) 25: 729, (1966).

(6) Sokolowsky et al., J. Am. Chem., Soc. 86: 1212, (1964).

(7) Tabachnick et al., J. Biol. Chem. 234, No. 7, p. 1726, (1959).

(8) Crampton et al., Proc. Soc. Exper. Biol. & Med. 80: 448, (1952).

(9) Goodfriend et al., Science 144, p. 1344 (1964).

(10) Sela et al., J. Am. Chem. Soc., 78, p. 746, (1955).

(11) Cinader et al., Brit. of Exp. Pathol. 36, p. 515, (1955).

(12) Phillips et al, J. of Biol. Chem. 240 (2), pp. 699-704, (1965).

(13) Bahl, J. of Biol. Chem., 244, p. 575, (1969).

Methods for preparing the modified polypeptides of this invention alsoinclude the following.

In one preferred modification approach, the polypeptide fraction, forexample, structure (XII), is activated first following which it isconjugated with a carrier, for example the influenza subunit describedabove, tetanus toxoid or Flagellin. An activating reagent may beutilized which exhibits differing functionality at its ends and bychoice of reaction conditions, these end components can be made to reactselectively. For example, the following activators A and B, having amaleiimido group and a substituted acid group, may be provided: ##STR1##where X is a non-reacting group made up of a substituted, orunsubstituted phenyl or C₁ -C₁₀ alkalene moiety, or a combinationthereof. In this regard, the moiety substituted on the phenyl should benon-interfering as is the remainder of the "X" grouping. X may, interalia, be selected from the following: ##STR2##

The maleiimido grouping of the above reagents will react with sulfhydryl(SH) groups in the polypeptide fragments under conditions whereby theopposite end (active ester end) of the reagent does not react with theamino groups present in the fragment sequences. Thus, for example,polypeptide fragments such as structure (XII), containing a Cys aminoacid and hence, as SH group react as follows: ##STR3## Following theabove, upon adjusting the pH to a slightly alkaline condition, e.g. 8,and adding the carrier protein accomplishes the following conjugation:##STR4##

Alternately, a carrier protein such as the above-noted Flagellin whichdoes not contain SH groups, but does contain NH₂ groups, may first betreated with activator A or B at pH 7 or lower at the active ester end,giving: ##STR5## Following the above, the activated carrier is reactedwith a polypeptide fragment containing an SH group to derive a productsimilar to that discussed immediately above.

Should the polypeptide fragment not contain an SH group, e.g. Structures(II), (III), (VI) and (VII), such structures can be modified first tointroduce such a grouping by standard methods such as"thiolactonization", following which they are conjugated utilizing theabove-discussed selective bi-functional reagents. For a more detaileddescription of these reagents, reference is made to the followingpublications:

O. Keller and J. Rudinger, Helv. Chim. Acta 58, 531-541 (1975).

W. Trommer, H. Kolkenbrock & G. Pfleiderer, Hoppe-Seyler's Z. Physiol.Chem., 356, 1455-1458 (1975).

Further description of the preferred embodiments of the above-describedutilization of bi-functional reagents is provided hereinbelow atExamples XXVII and XXVIII.

As an alternate approach to the utilization of the maleiimido groupreagents discussed above, an alkylation step may be used to causeconjugation. Conditions can be chosen such that in the presence of aminogroups, essentially only SH groups will be alkylated. With thisapproach, a generalization of the reactions carried out may be expressedas follows: ##STR6##

With this approach, the larger molecule carrier, e.g. Flagellin, tetanustoxiod or the influenza subunit described herein is first modified byreaction of a fraction of its amino groups with an active ester ofchloro, dichloro, bromo or iodo acetic acid, such as: ##STR7## and thismodified carrier is then reacted with the sulfhydryl group in apolypeptide fraction, or a polypeptide fraction which has been modifiedto contain the SH group (e.g. thiolactonization) if it does not alreadyhave such a group. Such modification is described in Example XXV below.The present approach produces a thio ether linkage by alkylation of afree thiol (sulfhydryl group).

With the instant procedure, the roles of the fragment and carrier may bereversed, the fragment being modified to contain the halomethylalkylating group which would then react with sulfhydryl groups in thecarrier, or a carrier suitably modified to exhibit a sulfhydryl group.More description of this selective alkylation of sulfhydryl groups isprovided in conjunction with Example XXX below.

It may be seen from an observation of the formulae of Structures (IV),(V), (IX), (X), (XI), (XII), (XIII) and (XIV) that a Cys amino acid,which in a reduced state provides an SH reactive group, is located ateither the C terminal or N terminal of the peptide structure. Thislocation permits the peptide to be chemically linked to carriermolecules at either terminus. And some Structures (XIV), (X), (IX), (X),(IV) have a six-Prolene spacer chain (Pro)₆ between the Cys residue andthe remainder of the peptide sequence. This latter arrangement providesa chemical spacer between the coupled carrier and the sequencesrepresenting a fragment of the natural hormone. A six-Prolene spacer canbe added as a side chain spacer, for example at position 122 (Lys) inStructure (II), by initially adding an SH group (thiolactonization) tothe free or unblocked epsilon amino group on this (Lys) residue, as setout in Example XXIX below. Then, utilizing the activator A,B above inwhich the component "X" is a chain of six Prolene amino acids,conjugation can be carried out. In the latter case, a spacer is providedbetween the carrier and peptide linked at an intermediate site, forexample at position 122 in structure II. In the former case, only thespace represented by conjugating reagent links the carrier and peptide.

Modifying groups, such as hemocyanin from Keyhole limpet, containingfree amino groups, are prepared in buffer solution such as phosphatebuffer, in sodium chloride solution at a pH of 6-8. To this solution,tolylene diisocyanate (T.D.I.C.) reagent diluted from about 1-10 toabout 1-40 times with dioxane, is added to the modifying group. Thegeneral procedure was disclosed by Singer and Schick, J. Biophysical andBiochem. Cytology 9:519 (1961). The amount of T.D.I.C. added may rangefrom 0.075 to 1,000 molar equivalents of the modifier used. The reactionmay be carried out at about -5° to about +10° C., preferably 0° to 4°C., for about 1/2 to 2 hours. Any excess T.D.I.C. may be removed bycentrifugation. The precipitate may be washed with the above-mentionedphosphate buffer and the supernatants combined.

This activated modifying group solution may then be combined with thehormonal or non-hormonal polypeptide to be conjugated. Polypeptide isdissolved in the same phospate buffer (5-30 mg/ml) and the volume ofmodifier and polypeptide combined according to the molar ratio of thetwo desired in the conjugate. Combined solutions are reacted at 30°-50°C., preferably 35°-40° C., for 3-6 hours.

Separation of modified polypeptide and free unconjugated polypeptide maybe accomplished by conventional techniques, such as gel filtration.

Picogram amounts of I¹²⁵ labeled polypeptide may be added as a tracer tothe reaction mixture at the time of conjugation, and a quantity ofpolypeptide conjugated to modifying groups (molar ratio) may bedetermined by the amount of radioactivity recovered.

Included in the methods for modifying the hormones, non-hormonalproteins and their fragments (unmodified polypeptides) are conjugationby use of water-soluble carbodiimide. The amino groups of the unmodifiedpolypeptide are first preferably protected by acetylation. This(acetylated) unmodified polypeptide is then conjugated to modifier, suchas natural protein modifier, e.g. hemocyanin from Keyhole limpet,homologous serum albumin, and the like, or Dextrans, Ficolls, orpolytyrosine, preferably in the presence of guanidine such as guanidineHCl, using 10-ethyl-3 (3-dimethylamino propyl) carbodiimide asactivating agent. This method is generally disclosed by Hoare andKoshland, Jr., J. of Biological Chemistry 242:2447 (1967). In theinstance where Ficoll 70 is used, it is preferred that it be firsttreated with ethylene diamine so as to render the final coupling moreefficient. This treatment with ethylene diamine may be performed insolvent such as saline and dioxane at about room tempreature and a pH ofabout 9-12, preferably 10-11 for about 1/4 to about 2 hours. Theconjugation itself between the unmodified polypeptide and the modifiermay be performed in solvent such as glycine methyl ester whilemaintaining the pH at about 4-5, preferably about 4.5-4.8. Thetemperature of reaction is conveniently about room temperature and thereaction may be allowed to proceed for about 2-8 hours, preferably 5hours. The resulting modified polypeptide with which this invention isconcerned may be purified by conventional techniques, such as columnchromatography.

The immunogenic substances for this invention may also be provided bypolymerization of unmodified polypeptide using bifunctional imidoester.The imidoester, such as dimethyl adipimidate, dimethyl suberimidate anddiethyl malonimidate, may be used to form the polymer in a mannersimilar to the generally described methods of Hartman and Wold; Biochem.6:2439 (1967). The polymerization may take place conveniently at roomtemperature in aqueous solvent at a pH of about 9-12, preferably about10-11, over a period of 1/4-2 hours.

Said immunogenic substances may also be prepared by dimerization througha disulfide bond formed by oxidation of the thiol group on a Cys-residueusing iodosobenzoic acid and methods corresponding to known methods,such as room temperature reaction for about 10-40 minutes.

Modified polypeptides may also be prepared using glutaric dialdehyde asconjugating agent. According to a theory proposed by Richards andKnowles [J. Mol. Biol. 37:231 (1968)], commercial glutaric dialdehydecontains virtually no free glutaric dialdehyde, but rather consists of avery complex mixture of polymers rich in α, β-unsaturated aldehydes.Upon reaction with natural protein modifiers such as homologous serumalbumins, these polymers form a stable bond through the free aminogroup, leaving aldehyde groups free. This intermediate product thenreacts with unmodified polypeptide in the presence of alkali metalborohydride, such as sodium borohydride. This intermediate is formed atpH 7-10, preferably 8-9, at about room tempreature. The modifiedpolypeptide is also conveniently obtained at about room temperatureafter about 1/4-2 hours' reaction time. The resulting product isrecovered in pure form by conventional techniques, such as gelfiltration, dialysis and lyophilization.

Polymerized sugar modifiers such as Ficoll 70 or Dextran T 70 may alsobe prepared for conjugation by treatment with a cyanuric halide such ascyanuric chloride to form a dihalotriazinyl adduct. The process may beperformed in solvent such as dimethylformamide at about 0°-20° C.,preferably 10°-15° C., for about 1/2-4 hours. The resulting intermediateproduct may then be dialyzed until essentially halogen ion free, andlyophilized and treated with unmodified polypeptides at pH 8-11,preferably about 9-10, for about 1/2-12 hours at about 15°-35° C.,conveniently at room temperature. The resulting modified polypeptide maybe recovered as indicated above.

Said polymerized sugar modifiers may also be treated with alkali metalperiodate, such as sodium periodate, at a pH of 3-6 at about 30°-60° C.for about 1/2-4 hours, and the resulting intermediate conjugated withunmodified polypeptide at a pH of about 7-11, preferably about 8-10, forabout 1/4 to about 2 hours at a temperature of about 15°-80° C.,preferably 20°-60° C. The resulting immunogenic substance according tothis invention may be separated as indicated previously.

The modifying groups may vary in chemistry and number for any givenpolypeptide structure. However, they will attach to only certain aminoacid moieties. In particular, when modifying with diazo groups they willchemically bond to only the histidine, arginine, tyrosine and lysinemoieties or sites. Other modifying groups will bond to peptide moleculesat different sites and in different numbers. Consequently, dependingupon the size and chemical make-up of a particular modified polypeptidedesired, one skilled in the art will readily be able to calculate themaximum possible number of modifying groups associable with apolypeptide. It is also recognized that several modifying groups mayattach themselves to each other which in turn attach to a single aminoacid moiety, but as used herein, reference to a number of modifyinggroups means the number of reaction sites to which a modifier has beenattached.

As indicated above, a theory leading to this invention was that thechemical modification of an essential reproductive hormone would alterit such that it would exhibit antigenic properties so that when injectedinto an animal (including humans) it would cause the formation ofantibodies which in turn would not only react to the injected modifiedhormone but also to the natural unmodified endogenous hormone as well.With this theory in mind, reproductive hormones of various species weremodified and tested in baboons. The results illustrated that modifiedhormones of unrelated species do not produce the desired results,whereas modified hormones of the same or closely related species doproduce the desired results. It will accordingly be clear that thepolypeptide to be modified should be so related to the endogenoushormone or non-hormonal protein as to be either from the same animalspecies or be the immunological equivalent thereof as modified.

Additional experiments were conducted to test the validity of thisconcept in humans, i.e. modified human reproductive hormones injectedinto humans. Collectively, the results prove the conclusion drawn fromthe experiments with the baboons, namely, isoantigenic immunizationusing modified human reproductive hormones does produce contraception orinterruption of gestation. Detailed examples which follow illustratethis result.

It is known that fragments of endogenous hormones exhibit essentially noantigenic properties. However, should a large enough fragment of anendogenous hormone be slightly modified as indicated above, thenantibodies will be formed which will react in the same way as if themodification is on a whole hormone, provided the large fragment issufficiently distinctive in chemical and physical make-up as to berecognized as a specific part of the whole.

Whether the hormone or specific fragment thereof is naturally occurringor is a synthetic product is clearly immaterial. A synthetic hormonemolecule will perform the same function as the naturally occurring one,being equivalent for the purpose of this invention. In this connection,it will be noted that natural substances with which this invention isconcerned possess carbohydrate moieties attached at certain sitesthereof whereas the contemplated corresponding synthetic polypeptides donot. Nevertheless, for the purpose of the instant specification andclaims, the synthetic and natural polypeptides are treated asequivalents and both are intended to be embraced by this invention.Reference in the above regard is made to Table No. 3 herein as read inconjunction with Example XXIX.

Thus, where the word "hormone" or "hormone molecule" is used herein, theword "synthetic" may be added before "hormone" without changing themeaning of the discussion. Similarly, the word, "fragment" may beinserted after "hormone" or "molecule" without changing the meaning,whether or not "synthetic" has been inserted before "hormone".

Throughout the above specification, the term "modified" has beenutilized in referring to the chemical reaction by which the foreignmolecules become chemically attached to specific sites on the usuallymuch larger polypeptide molecule. Although specific mechanisms by whichthis is accomplished are described herein in detail, other appropriatemechanisms may be used if desired. It is clear that the modifier, i.e.,the substance which modifies the concerned protein, can be a physicallylarger molecule or fragment thereof than the molecule or fragment whichit modifies. As noted above, such large molecules are deemed herein tobe "carriers". Clearly, physical size of the fragment is not alwayscritical; the criterion for effectiveness being that the body reactiongenerate antibodies in sufficient quanta and specific to the targetedhormone or endogenous substance.

The modified polypeptides of this invention may be administeredparenterally to the animals to be protected, preferably with apharmaceutically acceptable injectable vehicle. They may be administeredin conventional vehicles with other standard adjuvants, as may bedesirable, in the form of injectable solutions or suspensions. Asindicated earlier, the adjuvant serves as a substance which will elevatetotal immune response in the course of the immunization procedure.Lipasomes have been suggested as suitable adjuvants. The soluble saltsof aluminum, that is, aluminum phosphate or aluminum hydroxide, havebeen utilized as adjuvants in routine clinical applications in man.Bacterial endotoxins or endotoxoids have been used as adjuvants as wellas polynucleotides and polyelectrolytes and water soluble adjuvants suchas muramyl dipeptides. The adjuvants developed by Freund have long beenknown by investigators, however, the use thereof is limited to non-humanexperimental procedures by virtue of a variety of side effects evoked.The preferred mode of administration of the entire vaccine isintramuscular.

The amount of modified polypeptide to be administered will varydepending upon various factors, including the condition being treatedand its severity. However, in general, unit doses of 0.1-50 mg in largemammals administered one to five times at intervals of one to five weeksprovide satisfactory results. Primary immunization may also be followedby "booster" immunization at one to twelve month intervals.

DESCRIPTION OF THE PREFERRED EMBODIMENTS EXAMPLE I

Adult female baboons were studied for at least one menstrual cycle forpatterns of urinary estrogens, plasma, progestin, and in some casesurinary LH. Only those animals displaying normal patterns of thesehormones were immunized. The criteria for normality and the proceduresfor housing animals are well known and will not be described.

Gonadotropin Preparations

Human Luteinizing Hormone (HLH)--partially purified preparation fromhuman pituitaries with a biological potency of 2.5 units per mg.(NIH-LH-SI).

Human Follicle Stimulating Hormone (HFSH)--a partially purifiedpreparation from human pituitaries with a biological potency of 86 unitsper mg. (NIH-FSH-SI).

Human Chorionic Gonadotropin (HCG)--a highly purified preparation fromhuman pregnancy urine with biological potency of 13,200 IU/mg. (2ndIRP-HCG).

Monkey Luteinizing Hormone (MLH)--a crude preparation from rhesus monkeypituitaries with a biological potency of 0.75 units per mg. (NIH-LH-SI).

Ovine Luteinizing Hormone (OLH) (NIH-LH-S5).

Baboon Luteinizing Hormone (BLH)--partially purified baboon pituitarypreparation with a biological potency of 1.1 units per mg. (NIH-LH-S1).

All preparations, excepting the OLH, were prepared in the inventor'slaboratory. LH and HCG biological activity was determined by the ovarianascorbic acid depletion test and the FSH preparation assayed by theovarian augmentation assay.

Hormones were altered as antigens by coupling with a hapten in varyingratios of hapten to hormone as described by Cinader et al., supra. Forconvenience, the Cinader process is discussed herein although Phillips,supra, may provide a more stable bond under certain circumstances. Inthis procedure, the protein hormone serves as a carrier and the haptenis coupled to it by diazo bonds. Although a variety of hapten groupswere coupled to different hormones, the same basic procedure was usedfor any combination. Fifteen to thirty-five haptenic groups per hormonemolecule were found most useful for preparing immunizing antigens. Thebasic reaction consisted of diazotizing the hapten (sulfanilic acid) byadding it to a solution of 0.11 N HCl and then slowly adding thissolution dropwise to a 1 percent solution of NaNO₂ with constantstirring at 4° C. Diazotization was considered complete when free HNO₂was detected in the reaction mixture. Although the above reaction wasaccomplished at 4° C., optimum temperatures for the reaction normallyare about 0°-6° C., although 4° C. is preferred.

The hapten-protein coupling was performed by dissolving the proteinhormone in an alkaline buffer, pH 8.0. The diazotized hapten was addedslowly to the hormone solution with continuous stirring at 4° C. The pHof the reaction was constantly monitored and kept near 8.0. After allthe hapten was added, the pH was finally adjusted to 8.0, stirred for1-2 hours and allowed to stand at 4° overnight. The mixture wasthoroughly dialyzed for 6-8 days against distilled water to removeunreacted hapten.

Although the number of diazo groups per hormone molecule could beregulated by the number of moles of hapten and hormone reacted, aparallel control experiment with S³⁵ labelled sulfanilic acid toevaluate the precise composition of the hapten-protein samples wasperformed with each diazotization. The same hormone preparation to beused for immunization was used in the control experiment. After thereaction was completed, an aliquot was taken from the reaction mixtureand the remainder thoroughly dialyzed. Equal volumes of the dialyzed andundialyzed solutions were counted by liquid scintillation. By comparingthe counts of the dialyzed and undialyzed samples, the moles of haptencoupled to each mole of hormone was calculated since the unreactedhapten was removed by dialysis. For this calculation, a molecular weightof 30,000 was assumed for all gonadotropin preparations.

Following dialysis, hapten-hormones were lyophilized and stored at 4° C.Diazo-HCG (35 groups/molecule) and HLH (26 groups/molecule) werebioassayed by the ovarian ascorbic acid depletion method and found toretain 62 and 85 percent respectively of the activity of the unalteredhormones from which they were derived. None of the other hormones wereassayed for biological activity.

Immunization Procedures

Female baboons received their initial immunization on days 3-5 of themenstrual cycle and the second and third injections one week apart. Thefourth injection was given 2-3 weeks after the third. A few animalsreceived a fifth injection at 70-80 days after the first injections. Allantigens were administered subcutaneously in a suspension of mannidemanoleate or peanut oil. Doses of antigens for each injection variedbetween 3 and 5 mg. Injection sites were inspected daily for 5 daysafter each immunization for local reactions.

Monitoring Effects of Immunization.

Daily 24-hour urine specimens and frequent serum samples were collectedduring at least one menstrual cycle prior to immunizations and followingimmunizations until the effects of treatment were assessed. Urinary LH,urinary estrogens and plasma progestins were measured. Antibodies weredetected in post-immunization serum samples by reacting 0.2 ml. of a1:1000 dilution of serum in phosphate-buffered saline (pH 7.4) 0.5percent normal baboon serum with 250 pg of 1¹³¹ labelled hormone. Serawere reacted with both the unaltered immunizing hormone and unalteredbaboon LH for antibody detection. A purified baboon LH preparation(1.9×NIH-LH-S1) was used as a tracer antigen. Antigen-antibody complexeswere precipitated with ovine anti-baboon gamma globulin after a 24-hr.incubation at 4° C. Antibody levels were expressed as pg of labelledhormone bound. Significant antibody levels were considered to be thosethat would bind 5.0 pg or more of the 1¹³¹ labelled antigen.

Antisera were fractionated by gel filtration of Sephadex G-200 accordingto the procedure of Fahey and Terry (at p. 36, Experimental Immunology,F. A. Davis Co., Philadelphia, Pa., 1967, incorporated by reference tothe extent necessary to understand the invention) to determine theproportion of IgM and IgG antibodies in the baboon sera. Since the IgGfraction in this procedure contained a portion of IgA and IgDantibodies, only IgM and total titers were determined. The IgM fractionfrom the column was reacted with 1¹³¹ hormones and the binding capacitydetermined. The volumes of the fractionated sera were adjusted so thatantibody levels would be comparable to those of whole serum.

Antibody Production

No significant reactions were observed at the site of injectionfollowing any immunization. On 4 occasions, a slight induration (2-3 cmin diameter) was seen when mannide manoleate was used as a vehicle butthe redness and swelling disappeared within 4-5 days. Antibodies weredetected against the immunizing antigen within 3-5 weeks in all animals.The extent, duration and cross reactivity of these antibodies isrecorded. Generally speaking, higher levels were observed toheterologous gonadotropin immunization than to homologous ones.

The cross-reactivity of induced antibodies with baboon LH was studied oneach animal. Cross-reactivity of antisera at peak levels was recorded.Although relatively high antibody activity against human LH and HCG wereseen, relatively little reaction with baboon LH occurred. Anintermediate cross-reaction was noted with anti-ovine LH and a highdegree of cross-reactivity was seen with anti-monkey LH. Diazo-human FSHwas weakly antigenic in the baboon. The duration of antibody productionwas generally longer with the human and sheep gonadotropin immunizationthan with those of monkey or baboon origin.

Peak antibody levels usually occurred at the time when the antibodieshad shifted to principally the IgG type. Early antibodies had a largerproportion of IgM type and were generally more cross-reactive withbaboon LH. The change in the proportion of the total antibody populationthat was IgM was recorded from the time antibodies were first detected.Significant cross-reactivity to baboon LH was observed in anti-humangonadotropins when IgM was abundant but dropped sharply as the antiserashifted to nearly all IgG. This drop in cross-reactivity did not occurwith monkey and baboon immunizations. Again, the ovine LH immunizationproduced an intermediate change in reactivity with the shift from IgM toIgG.

Effects on the Menstrual Cycle

The effects of immunization upon the event of the menstrual cycle weredetermined by observing changes in sex skin turgescence and levels ofpituitary and/or ovarian hormones. Based on these parameters, the delayor retardation of ovulation from the expected time, as judged by thecontrol cycle, was calculated. One animal immunized with HCG had nointerruption in ovulation and another immunized with HFSH was delayedfor only one cycle. Two animals injected with HLH and two injected withHCG had ovulation delays equivalent to two menstrual cycles. A thirdanimal immunized with HLH was delayed a calculated 86 days. Ovine LHimmunizations produced an 88 day delay in ovulation.

Immunizations with diazo-monkey or baboon LH resulted in longerdisruption of the menstrual cycle. Calculated delays in ovulation forthe two animals receiving monkey LH was 146 and 122 days whereas theanimals receiving altered baboon LH were retarded from ovulation 224 and210 days.

Effects on specific hormone patterns following immunization with HLH inone animal were recorded. The interval between menses was considered torepresent a "cycle". Urinary estrogens and plasma progestin patternsindicated that no ovulation occurred during the cycle of immunizationwhich was 85 days in duration. Urinary estrogens were elevated duringtreatment but did not reflect a typical pattern. Plasma progestins werenot elevated until about day 19 of the first post-treatment cycle.Patterns of both estrogens and progestins were within normal limitsduring the second post-treatment cycle. Antibody levels were elevatedfrom about day 35 of the treatment cycle until 289 days from the firstdetection of antibodies. An LH assay was not available when this animalwas studied and no data on plasma or urinary levels of this hormone wasobtained.

Hormonal patterns following an immunization with diazobaboon LH wererecorded. In this animal, antibody levels were lower and persisted, ingeneral, for a shorter period than did immunizations with humangonadotropins. During the treatment cycle, levels of urinary estrogensand plasma progestins followed a normal pattern but were quantitativelylower than normal. Urinary LH patterns fluctuated markedly due to theinjections of diazo-LH during this period. No conclusive evidence ofovulation was obtained for the treatment cycle. The first post-treatmentcycle lasted 246 days. During this cycle urinary LH and estrogens wereelevated on days 35-41 but there was no subsequent elevation in plasmaprogestins that would indicate ovulation had occurred. Following day 42of this cycle, there was no significant elevation in any of the threehormone levels until day 231 when significant elevations of urinaryestrogens and LH occurred. These rises were followed 3 days later by anelevation in plasma progestins indicating the presence of a functioningcorpus luteum. A second post-treatment menstrual cycle was of normalduration and the endocrine patterns were normal.

Antibodies to unaltered baboon LH attained maximum levels by about day70 of the post-treatment cycle and remained relatively constant untilday 190 when a steady decline was observed. By day 215 of this cycle,antibody levels were barely detectable. Approximately 16 days after thistime, a peak of LH commensurate with a normal midcycle elevation wasobserved. From this point the animal appeared to have the normalfunction of the pituitary-ovarian axis. Hormonal patterns in animalswith other heterologous gonadotropin immunizations were similar toanimal receiving HLH and other animals receiving monkey or baboon LHwere similar in response to animal receiving baboon LH.

These results in baboons indicated that the modification of areproductive hormone, by the procedures outlined, did render itantigenic and the antibodies thus formed did neutralize naturalendogenous hormones if the natural hormone was obtained from the speciesreceiving the immunizations with modified hormone.

EXAMPLE II

HCG is a hormone naturally present only in pregnant women with theexception that an entity at least analogous thereto has been found to bepresent in humans in conjunction with neoplasms. HCG is alsocommercially available. Human LH is immunologically and biologicallyidentical to HCG, even though there are chemical differences. Since theyare biologically identical and HCG is readily available from commercialsources it was presumed that the effectiveness of this immunologicalprocedure could be evaluated by injecting modified HCG into non-pregnantwomen and monitoring the blood levels of LH. Antibodies formed willneutralize both the LH and the modified HCG. Reference in the aboveregard is made to the publications identified earlier herein.

Women have a pattern of LH levels; the level is substantially constantuntil the middle period between menstrual cycles, immediately prior toovulation; at that point of LH level rises greatly and helps induce theovulation. Monitoring the LH level and the antibody level will show thatthe procedure used did or did not cause the production of antibodiescapable of neutralizing the endogenous reproductive hormone, namely LH.

A woman aged 27 years was selected for study. Hormone was obtained,purified and modified. The modified human hormone (HCG) was injectedinto the subject. It is well known that antibodies to HCG reactidentically to LH as well as HCG. The effect of the immunization wasevaluated, principally by monitoring blood levels of LH. Finally theresults were evaluated.

Preparation of Hormone

Clinical grade HCG derived from pregnancy urine was obtained from theVitamerican Corp., Little Falls, N.J. This material has an immunologicalpotency of 2600 IU/mg. Contaminants were detected in this preparation.Purification consisted of chromatography and elution. Fractions weredialyzed and lyophylized. The most potent fraction containedapproximately 7600 IU/mg., however, it was heterogenous onpoloyacrylamide gel electrophoresis.

The fraction was further purified by gel filtration. The elution profilerevealed two major protein peaks. The most potent HCG was found in thefirst peak and had an immunological potency of 13,670 IU per mg. Thisfraction was subjected to polyacrylamide gel electrophoresis. Furtherpurification by gel filtration showed no evidence of heterogeneity ofthe HCG at this stage. Consequently, materials for study were processedaccording to the above procedure.

The contamination of this purified HCG was tested with I¹³¹ used foridentification and a sample was reacted with antisera against severalproteins offering potential contamination. Those proteins were folliclestimulating hormone, human growth hormone, whole human serum, humanalbumin, transferin, alpha one globulin, alpha two globulin andorosomucoid. No detectable binding of the purified HCG was observed withany antisera at a dulution of 1:50 of each. These negative results,calculated against potential binding of the respective proteins,indicated that contamination with any was less than 0.005 percent.

Alteration of Hormone

Hormone was altered by coupling with a hapten (sulfanilazo). This methodcouples the hapten molecules to the protein via the amino group of theliphatic or aromatic portion of the hapten. The number of haptenmolecules coupled to each HCG molecule (Ha-HCG) can be regulated and forthis study, forty haptenic groups per HCG molecule were used forpreparing the immunizing antigen.

Following the hapten-coupling process, the Ha-HCG was sterilized andtested.

Subject

The subject was multiparous and had terminated her reproductivecapabilities by prior elective bilateral salpingectomy. She was in goodhealth and had regular cyclic menstruation. She underwent completehistory, physical exmination and laboratory evaluation including bloodcount, urinalysis, latex fixation and Papanicolau smear. She had nohistory of allergy.

To demonstrate normal functioning of the pituitary-ovarian axis prior toimmunization, blood samples were obtained every other day from the firstday of menses for 10 days, then daily for 10 days and finally, everyother day until the next menses. Serum determination of FSH, LH,estrone, estradiol and progesterone were performed. These studiesindicated an ovulatory pattern.

Immunization Procedures

Ten mg. of the Ha-HCG antigen were dissolved in 1.0 ml. of saline andemulsified with an equal volume of oil. Prior to injection, scratchtests to antigen and vehicle were performed. Immunizations were begun inthe luteal phase of the treatment cycle to prevent superovulation fromthe administered HCG. Four injections at two week intervals were givento the subject. The first two of these were administered in oilsubcutaneously (1.0 ml. in each upper arm); the final two injectionswere given in saline only via the intradermal route. Following eachinjection, blood pressure readings were taken and the subject observedfor allergic reactions.

Monitoring Effects of Immunizations

Blood samples were collected at weekly intervals beginning two weeksafter the initial injection to test for the presence of humoral andcellular antibodies. Following completion of the immunization schedule,blood samples were collected in the same manner as in the control cycleto assess effects of immunization on hormonal patterns of the menstrualcycle. Since antibodies to HCG react identically to LH as with HCG, LHwas monitored as an index of effectiveness of the procedure. A thirdcycle was similarly studied six months after initial immunization. Uponcompletion of the study, physical and pelvic examinations and laboratoryevaluations were repeated.

Serum samples from the control and post-treatment cycles were assayedfor FSH, LH, estrone, estradiol and progesterone.

The subject was tested for delayed hypertensivity before immunizationand at two week intervals until the injection schedule was completed byan in vitro lymphocyte transformation test.

Results

Temporal relationships of serum pituitary and gonadal hormones in thecontrol cycles of the subject were recorded. Antibody titers to HCG weredetected in the subject after two injections. Menses occurred at regularintervals during the immunizations.

Following the initial injection in mannide manoleate, some itching andswelling at the injection site occurred. Subsequent intradermalinjections in saline produced no reactions and it was concluded that thelocal reactions were induced by the mannide manoleate. Lymphocytetransformation tests on plasma samples were negative.

In the post-treatment cycle, baseline follicular and luteal phase LHlevels were not noticeably changed in the subject. Very small midcycleelevation in LH levels were observed as compared to the normal largeincreases. FSH patterns in the post-treatment cycle were normal. Thisindicated that the antibodies were neutralizing the action of endogenousLH.

The subject showed an ovulatory progesterone pattern but attainedrelatively high antibody titers to LH and HCG after only two injectionsof Ha-HCG.

The subject was studied during another cycle approximately six monthsfrom the first immunization. Significant antibody titers were found. LHpatterns indicated a small midcycle elevation. FSH patterns wereessentially normal. Thus, the specificity of anti-HCG antibodies to LHwas shown but not to FSH.

EXAMPLE III

Another woman aged 29 years was selected for further study. Hormone wasobtained, purified, and modified as in Example II. This modified hormonewas injected into this subject in the same way as in Example II. Thesubject was monitored and tested as in Example II.

The results were similar to the results found in Example II except that(1) the levels of estrone and estradiol were substantially normal, (2)the subject acquired significant antibody titers late in thepost-immunization cycle, and (3) in the cycle studied after six monthsthis subject showed no significant midcycle elevation in LH patters.

EXAMPLE IV

Another woman aged 29 years was selected for further study. Hormone wasobtained and purified and modified as in Example II. This modifiedhormone was injected into this subject in the same way as in Example II.The subject was monitored and tested as in Example II.

The results were similar to the results found in Example II except that(1) baseline follicular and luteal phase LH levels were noticeablydepressed in the post-treatment cycle, (2) no midcycle elevations wereobserved in LH, (3) estrone levels were elevated during the follicularphase of the post-immunization cycle, and (4) during the six-monthsstudy there was no significant midcycle elevation in LH patterns.

EXAMPLE V

Another woman aged 35 years was selected for further study. Hormone wasobtained, purified, and modified as in Example II. This modified hormonewas injected into this subject in the same way as in Example II. Thesubject was monitored and tested as in Example II.

The results were similar to the results found in Example II except that(1) baseline follicular and luteal phase LH levels were noticeablydepressed in the post-treatment cycle, (2) a very small midcycleelevation of LH was observed, (3) levels of FSH patterns in thepost-treatment cycle were depressed, and (4) levels of both estrone andestradiol were reduced, during the follicular phase of thepost-immunization.

EXAMPLE VI

Another woman aged 28 years was selected for further study. Hormone wasobtained, purified, and modified as in Example II. This modified hormonewas injected into this subject in the same way as in Example II. Thesubject was monitored and tested as in Example II.

The results were similar to results found in Example II except that (1)baseline follicular and luteal phase LH levels were depressed in thepost-treatment cycle, (2) no peaks were observed in midcycle levels ofLH, (3) estrone levels appeared elevated in the follicular phase of thepost immunization cycle, and (4) LH patterns indicated no significantmidcycle elevation in the six-month post-immunization cycle.

EXAMPLE VII

Another woman aged 28 was selected for further study. Hormone wasobtained, purified, and modified as in Example II. This modified hormonewas injected into this subject in the same way as in Example II. Thesubject was monitored and tested as in Example II.

The results were similar to results found in Example II except that (1)antibody titers to HCG were not detected until after three injections,(2) baseline follicular and luteal phase LH levels were depressed in thepost-treatment cycle, (3) no peaks nor midcycle elevation in the LH wereobserved, (4) estrone levels were elevated during the follicular phase,and (5) no significant antibody titers were found in the six-monthcycle.

All the above examples show the practicality of injecting modifiedhormones for the purpose of neutralizing an endogenous reproductivehormone and thereby offering a procedure for the prevention ofconception or the disruption of gestation.

EXAMPLE VIII

Data obtained in earlier experiments and discussed in Examples I-VIIshowed that a modified natural reproductive hormone, when injected intoan animal of species from which it was derived, would produce antibodiesthat would neutralize the action of the unmodified endogenous naturalhormone in the body of the animal. Hormones used in Examples I-VII wereFSH, LH and HCG. New experiments were performed, based on thisknowledge, to identify another reproductive hormone (placental lactogen)that could be used in a similar fashion.

Preparation of Hormone

A purified preparation of placental lactogen was prepared from placentaeof baboons since it was intended to use modified placental lactogen toimmunize baboons. Placentae were extracted and purified on columnchromatograph according to previously published procedures. The puritywas tested by polyacrylamide gel, electrophoresis and byradioimmunoassay. The material obtained showed a high degree of purityon electrophoresis and radioimmunoassay showed no contamination withother placental hormones.

Hormone Modification and Immunizations

The baboon placental lactogen (BPL) was altered by coupling with thediazonium salt of sulfanilic acid as outlined for other hormones inExample I. The number of diazo molecules per BPL molecule in thisinstance was 15. Immunization procedures were also similar to thosedescribed in Example I for other hormones.

Results

Within 4-6 weeks after the first injection of diazo-BPL, antibody levelsto natural unmodified BPL in vitro were detected in 6 female baboons.Levels rose to a plateau within 8-10 weeks and remained there forseveral months. Hormonal measurements indicated that there were noeffects on the normal events of the menstrual cycle due to theimmunizations. Since BPL is normally secreted only in pregnancy, thiswas not a surprising observation.

All six females were mated with a male of proven fertility three times(once each in three different cycles during the fertile period).Pregnancy diagnosis by hormonal measurement was performed after eachmating. From the 18 matings, there were 13 conceptions as judged bypregnancy tests. The animals that were pregnant had menstrual bleeding7-12 days later than was expected for their normal menstrual cycles.Subsequent hormonal measurements confirmed that these 13 pregnancieswere terminated by abortions approximately one week after the time ofexpected menses.

These findings suggest that the antibodies formed in the animals bodyafter immunization had no effect on the nonpregnant menstrual cycle butwhen pregnancy was established, they neutralized the baboon placentallactogen in the baboon placenta and the result was abortion very earlyafter conception.

When in Examples I-VIII above Structures (I), (II), (III) are modifiedby use of diazosulfanilic acid, dinitrophenol, or S-acetomercaptosuccinic anhydride or Structures (II), (III) are modified byaddition of polytyrosine or polyalanine, according to known methods, theresults obtained shall be similar to those in said Examples.

Similarly, when FSH, somatomedian, growth hormone or angiotension II aremodified by use of diazosulfanilic acid or trinitrophenol, the resultsobtainable upon administration of the purified modified polypeptide intoa male or female human or animal would indicate the stimulation ofantibodies which neutralizes all or some of the modified polypeptide aswell as corresponding endogenous polypeptide.

EXAMPLE IX

The subjects used in the studies reported in the example are femalebaboons. All baboons were adults of reproductive age. A description ofsubjects and the conditions of experimentation have been described inExample I. The animals have been studied using highly purified betasubunits of HCG using a preparation with a biological activity of lessthan 1.0 IU/mg. Animals were immunized with 14-26 moles/mole ofpolypeptide of diazosulfanilic acid coupled subunits in mannidemanoleate.

Antibody levels were assessed by determining the binding of serumdilutions with I¹²⁵ labelled antigens. Cross-reactivity of antisera wasmeasured by direct binding of labelled antigens and by displacementradioimmunoassays. Antifertility effects in actively immunized animalswere tested by mating females with males of proven fertility. Effects inpregnant baboons passively immunized with either sheep or baboonanti-β-HCG were determined by monitoring serum levels of gonadotropinsand sex steroid hormones before and after immunizations.

Eight female baboons were immunized with the modified beta subunit ofHCG. Significant antibody levels were attained in all animals.

Baboon immunizations with the modified beta subunit of HCG resulted inhigh antibody levels reacting to HCG, human LH and baboon CG but not tobaboon LH. All animals remained ovulatory, however, no pregnanciesresulted from numerous matings with males of proven fertility. Passiveimmunization of non-immunized pregnant baboons with sheep anti-β-HCGserum produced abortions within 36-44 hours.

EXAMPLE X

Hemocyanin from Keyhole limpet (KLH) solution (7 mg/ml) in 0.05 M sodiumphosphate buffer in 0.2 M NaCl, pH 7.5, is prepared. Insoluble particlesare removed by centrifugation. To one ml of this solution, tolylenediisocyanate (T.D.I.C.) reagent is added (20 μl) diluted to 1/30 withdioxane, the amount being essentially the equivalent of the moles oflysyl residues in the KLH molecules. After 40 minutes at 0° C., theT.D.I.C. activated KLH solution is combined with 0.5 mg of syntheticβ-HCG peptide having the following structure:

Asp-His-Pro-Leu-Thr-Cys-Asp-Asp-Pro-Arg-Phe-Gln-Asp-Ser-Ser-Ser-Ser-Lys-Als-Pro-Pro-Pro-Ser-Leu-Pro-Ser-Pro-Ser-Arg-Leu-Pro-Gly-Pro-Pro-Asp-Thr-Pro-Ile-Leu-Pro-Gln-Ser-Leu-ProStructure (XV)

which is first dissolved in 25 μl of 0.05 M sodium phosphate buffer in0.2 M, NaCL, pH 7.5. The mixture is incubated at 37° C. for four hours.The resulting product is purified by gel filtration.

EXAMPLE XI

One g. of Ficoll 70 is dissolved in 1 ml each of normal saline and 2 Methylene diamine (adjusted to pH 10 with hydrochloric acid) solution.The solution is kept at room temperature in a water bath and stirredwith a magnetic stirrer. Cyanogen bromide, 4 g, dissolved in 8 ml ofdioxane, is added to the Ficoll 70 solution. The acidity of the mixtureis maintained at pH 10-10.5 for 8 minutes by adding drops of 2 N sodiumhydroxide solution. An additional 2 ml of 2 M ethylene diamine, pH 10,solution is added, and stirring at room temperature is continued for 30more minutes. The product is purified by passing it through a Bio-Gelp-60 column.

EXAMPLE XII

Two mg of the compound of Structure (II) containing picogram amount ofI¹²⁵ labeled adduct and KLH (1.6 mg) of dissolved in 1 ml. of 1.0 Mglycine methyl ester in 5 M guanidine hydrochloride. Ethyl dimethylaminopropylcarbodiimide (E.D.C.) 19.1 mg is added to this solution. Theacidity is adjusted to and maintained at pH 4.75 with 1 N HCl at roomtemperature for 5 hours. The KLH-peptide conjugate is purified bypassing it through a Bio-Gel p-60 2.2×28 cm column equilibrated with 0.2M NaCl.

EXAMPLE XIII

Solid bifunctional imidoester dihydrochloride (3 mole) is added in 2 mgportions at 5-minute intervals to a constantly stirred solution of 1mole of polypeptide of Structure (II) (1-20 mg/ml) in 0.1 M sodiumphosphate, pH 10.5, at room temperature. Sodium hydroxide 0.1 N is addedto maintain the acidity at pH 10.5. One hour after the addition of thediimidoester has been completed, a polymerized product according to thisinvention is obtained.

EXAMPLE XIV

To a 20 mg/ml solution of homologous serum albumin in 0.1 M boratebuffer, pH 8.5, 1000% mole excess of 25% aqueous solution of glutaricdialdehyde is added at room temperature. The excess dialdehyde isremoved by gel filtration in water using Bio-Gel p-2. The materialcollected at the void volume is lyophilized, and the dried product isredissolved in 0.1 M borate buffer, pH 8.5 (20 mg/ml), mixed with therequired amount of polypeptide of the following Structure:

Asp-Asp-Pro-Arg-Phe-Gln-Asp-Ser-Ser-Ser-Ser-Lys-Als-Pro-Pro-Pro-Ser-Leu-Pro-Ser-Pro-Ser-Arg-Leu-Pro-Gly-Pro-Pro-Asp-Thr-Pro-Ile-Leu-Pro-Gln-Ser-Leu-ProStructure (XVI)

(20 mg/ml) in the same buffer at room temperature. Twenty minutes later,sodium borohydride in 250 percent molar excess of polypeptide XVI isadded. The reaction is terminated after one hour. The conjugated productis purified by gel filtration on Bio-Gel p-60 column, dialyzed free ofsalt and lyophilized.

EXAMPLE XV

Ficoll 70 1 g, NaHCO₃ 500 mg, cyanuric chloride 3 g, H₂ O 20 ml, anddimethylformamide 80 ml., are stirred at temperature below 16° C. for 2hours. The product is dialyzed against distilled water until Cl-free,then lyophilized. A polypeptide of Structure (XV) (2 mg) containingminute quantity of I¹²⁵ -labeled analogue is incubated with 1 mg of thisproduct in 0.25 ml of 0.2 M sodium borate buffer, pH 9.5, for one hourat 20° C., and the product is recovered from a Bio-Gel p-60 2.2×28 cmcolumn.

When the above procedure is carried out and Dextran T 70 is used inplace of Ficoll 70, the corresponding modified polypeptide, usefulaccording to this disclosure, is obtained.

EXAMPLE XVI

Ficoll 70 1 g, NaIO₄ 1.2 g, and KCl 0.42 g are dissolved in 1.5 ml of 1M sodium acetate buffer, pH 4.5, and incubated at 37° C. for 1 hour.

Two mg (=588 μmoles) of polypeptide of Structure (XV) above mixed withminute quantity of I¹²⁵ -labeled analogue is incubated with 2 mg of theproduct obtained above in 0.3 ml of 0.2 M borate buffer, pH 9.5 at 55°C. for 1 hour. The reaction mixture is then chilled in an ice water bathand NaBH₄ 1 mg is then added into this solution. The reduction reactionis terminated by passing the product through a Bio-Gel p-60 2.2×28 cmcolumn equilibrated and eluted with 0.2 M NaCl.

EXAMPLE XVII

Numerous rabbits are immunized with a variety of synthetic peptidesconjugated to different modifying groups. Following two or threeimmunizations at 3-5 week intervals, sera from animals are assessed bydetermining their ability to bind in vitro to radiolabeled HCG. Thespecificity of this binding is studied by reacting the same sera againstsimilarly labeled other protein hormones, particularly, pituitary LH.Sera are further assessed by determining their ability to inhibit thebiological action of exogenously administered HCG in bioassay animals.Thus, the increase in uterine weight of the immature female rat inresponse to a prescribed dose of HCG is noted. The dose of HCG isadministered subcutaneously in saline in five injections over a threeday period and the animal is sacrificed for removal of the uterus on thefourth day. The weight of the uterus increases in dose reponse fashionto the hormone injections. When assessing the effects of antisera inthis response, varying quantities of test serum are administeredintraperitoneally separately from the subcutaneous injection of hormoneduring the assay. This procedure permits the antiserum to be absorbedrapidly into the rat's bloodstream and will permit interaction of itwith hormone when the latter likewise enters this fluid. If theantiserum is capable of reacting with the hormone in a manner preventingstimulation of the uterus, the antiserum is considered to be effectivefor biological inhibition of hormone action.

The frequency of animals showing a positive response to immunologicalbinding and neutralization of biological activity is presented in

EXAMPLE XVIII

Iodosobenzoic acid dissolved in a slight excess of 1 N potassiumhydroxide in 10% molar excess is added to the peptide of Structure (II)in phosphate buffer with normal saline at pH of 7.0. After thirtyminutes at room temperature, the product polypeptide dimer is purifiedby gel filtration.

EXAMPLE XIX

To an ice water bath cooled and vigorously stirred 0.23 ml. of bovinegamma globulin (10 mg/ml) in 0.05 M phosphate buffer with normal saline(PBS) pH 7.5, 50 μl of 1/10 T.D.I.C. in dioxane is added. After 40minutes, the excess T.D.I.C. is removed by centrifugation (0° C., 10minutes, 10,000 g) and the precipitate is washed twice with 0.1 ml. ofPBS. The combined supernatents are added to 7.7 mg. of the peptide ofstructure II dissolved in 0.8 ml. of PBS, pH 7.5. The mixture is stirredat room temperature for 10 minutes, then incubated at 37° C. for 4hours. The conjugate product is purified by dialysis.

EXAMPLE XX

BSA (10 mg/ml) in PBS solution (0.25 ml.) is treated with 50 μl of 1/10T.D.I.C. dioxane solution and conjugated to 7.5 mg. of synthetic β-HCGpeptide of Structure (III) in 0.8 ml. of PBS (pH 7.5) as in Example XIXto obtain the product.

EXAMPLE XXI

To an ice water bath cooled and vigorously stirred 0.6 ml. of β-HCGpeptide of Structure (III) (10 mg/ml) in phosphate buffered saline, pH7.5, is added 30 μl of 1/10 T.D.I.C. After 40 minutes, the excessT.D.I.C. is removed by centrifugation (10,000 g, 0° C., 10 minutes) andthe precipitate is washed twice with 0.1 ml. PBS. The combinedsupernatents are added to 3 mg. of poly (D, L-Lys-Als) dissolved in 0.3ml. of PBS. The mixture is incubated at 37° C. for 4 hours. The productis then dialyzed and lyophilized.

EXAMPLE XXII

The results set out in Table I provide further evidence of the broadapplicability of this invention as indicated previously in thisspecification.

Using standard methods of testing in rabbits, both immunological bindingresponse and neutralization of biological activity were established forthe modified polypeptides indicated with the result as set out in TableI.

EXAMPLE XXIII

Antigen was prepared by reacting a Diisocyanate (T.D.I.C.--see above)coupling reagent with carrier (tetanus toxoid), extracting excessreagent and incubating activated carrier with peptide Structure (II).Baboons were immunized with the antigen and the results of mating 4animals three times are shown in FIG. 1. The figure shows that from 12exposures (matings) one pregnancy resulted even though relatively lowlevels of immunity from the antigen were achieved. Non-immunized baboonsof the same colony had a fertility rate of approximately 85%.

EXAMPLE XXIV

Referring to FIG. 2, baboons were immunized initially with a betasubunit of HCG modified by diazotization in a manner similar to thatdescribed in conjunction with Example II. Following this initialadministration, the baboons were injected 21 and 42 days later withStructure (II) above having been modified by the same diazotizationprocess. FIG. 2 shows plots representing the levels of antibodiesgenerated in consequence of these administrations. Such quantities ofantibodies are expressed as micrograms of isotopically--labeled HCG thatwill bind each milliliter of serum from the baboons at specified daysafter the initial injection. The levels shown were maintained for aperiod of over one year.

                                      TABLE 1                                     __________________________________________________________________________    Frequency of Positive Antibody Responses to Various HCG                       Peptide-Conjugates                                                                                Number of Rabbits                                                                   Immunological                                                                           Neutralization of                         Peptide                                                                              Carrier      Immunized                                                                           Binding Responses                                                                       Biological Activity                       __________________________________________________________________________    35 amino acid                                                                 111-145                                                                              Bovine Gamma Globulin                                                                      10    10        6                                         Morgan et al                                                                         Keyhole Limpet                                                         Peptide II                                                                           Hemocyanin   10    5         *                                         31 amino acid                                                                 115-145                                                                              Poly-D-L-Alanine                                                                           10    9         5                                         Morgan et al                                                                         Bovine Serum Albumin                                                                       12    12        6                                         Peptide III                                                                   44 amino acid                                                                 105-148                                                                              Keyhole Limpet                                                                Hemocyanin   10    8         *                                         Peptide XV                                                                    Natural                                                                       109-145                                                                              Keyhole Limpet                                                         Keutman                                                                              Hemocyanin   10    10        *                                         Peptide XII                                                                   __________________________________________________________________________     *additional time needed for assessment                                   

Referring to Table 2, the results of breeding the two baboonsrepresented in FIG. 2 is revealed in tabular form. The table presentsthe results of mating these animals ten times over a period ofapproximately one year. These data suggest that the animals ovulated inevery cycle, however, no pregnancy was observed, as indicated by theanimal having a menstrual period at or before the expected timetherefor. While the results tabulated demonstrate the efficacy of theentire procedure, it was observed for the particular structure utilizedin the primary immunization, i.e. Structure (I), antibody crossreactivity with LH was observed. Such cross reactivity may be avoided bythe utilization of the fragment conjugation procedures set forth indetail hereinabove.

EXAMPLE XXV

The specificity of antibody response to a CG fragment-macromolecularcarrier is represented by the instant experiment. A 35 amino acidsequence [Structure (II), herein "synthetic peptide"] of the HCG betasubunit was conjugated with bovine gamma-globulin and administered to ababoon. Varying doses of each of these three hormones were tested fortheir ability to compete with I¹²⁵ -labeled synthetic peptide [structure(II)] bound to the antiserum. The results are set forth in FIG. 3. Notefrom the figure that Human LH was ineffective for displacement of tracerantigen at doses up to 2.5 IU (international units). Since HCG displacedantigen at a dose of 20 mIU, the cross-reactivity with HLH in this assaysystem was less than 0.8%. Baboon CG also displaced I¹²⁵ -labeledantigen in this assay and, based on biological potency of the twohormones, was about 20% as effective as HCG.

                  Table 2                                                         ______________________________________                                        Breeding of Immunized Baboons                                                 [Diazo-β-HCG presensitized]                                              Booster: Diazo-β-HCG-(111-145)                                           1                   2                                                                                     Pre-Mate                                          Pre-Mate Titer                                                                            Ovul.   Preg.   Titer  Ovul. Preg.                                ______________________________________                                        Mating No. 1                                                                   5.00       +       -       4.20   +     -                                    Mating No. 2                                                                   4.25       +       -       4.10   +     -                                    Mating No. 3                                                                   4.22       +       -       4.00   +     -                                    Mating No. 4                                                                   4.17       +       -       3.89   +     -                                    Mating No. 5                                                                   3.80       +       -       3.76   +     -                                    Mating No. 6                                                                   6.65       +       -       5.00   +     -                                    Mating No. 7                                                                   5.90       +       -       4.75   +     -                                    Mating No. 8                                                                   5.10       +       -       4.20   +     -                                    Mating No. 9                                                                   5.00       +       -       4.25   +     -                                    Mating No. 10                                                                  4.66       +       -       4.00   +     -                                    ______________________________________                                    

EXAMPLE XXVI

The following experiments were carried out to determine whether thecarbohydrate chains contained in the C-terminal 37 residues of β-HCGinfluence the immunogenicity of that peptide.

A peptide representing amino acid residues 109-145 of β-HCG was isolatedfrom a chymotriptic digest of reduced and carboxymethylated β-HCG byprocedures reported by Keutmann, H. T.; Williams, R. M., J. Biol. Chem.252, 5393-5397 (1977). This peptide is identified in Table 3 as P-1. Thepurity of the peptide was confirmed by amino acid and terminal end groupanalyses. A portion of the isolated peptide was treated with anhydroushydrofluoric acid (HF) to remove carbohydrate moieties and repurified bycolumn chromatography according to methods described by Sakakibara S. etal, Bull. Chem. Soc. Japan, 40, 2164-2167 (1967). This portion of theisolated peptide is identified in Table 3 as P-2. Complete removal ofthe sugar chains were confirmed by carbohydrate analysis; See Nelson,Norton, J. Biol. Chem. 153, 375-380 (1944). A third peptide with theamino acid sequence 109-145 of β-HCG was prepared synthetically usingthe solid state synthesis procedure of Tregear, G. W. et al., Biochem.16, 2817 (1977). This third peptide is identified in Table 3 as P-3.Highly purified HCG was used in all immunological experiments wherereference was made to intact HCG.

Preparation of Immunogens and Immunizations

Conjugates of the three peptides were prepared to keyhole-limpethemocyanin (KLH) using tolulene diisocyanate. A peptide-carrier ratio of4-6 peptides per 100,000 daltons of carrier was obtained for differentconjugates prepared according to amino acid analyses. Rabbits wereimmunized with conjugates by three multiple site intramuscularinjections of 1.0 mg. of conjugate in 0.5 ml. of saline emulsified withan equal volume of Freund's complete adjuvant. Injections were given at3 week intervals and weekly blood samples were collected from 3-20 weeksof immunization.

Evaluation of Antisera

Antisera to all conjugates were monitored for antibody levels byreacting dilutions of sera with I¹²⁵ labeled HCG (chloramine T method)at 4° C. for 5 days and precipitating immune complexes with sheepanti-rabbit gamma globulin serum. Antibody levels were determined byassessing dilution curves in which a linear correlation between dilutionand binding of labelled antigen at equilibrium occurred. At least 3points in each curve were used in calculating levels. These levels wereexpressed as μg. HCG bound per ml. of undiluted serum calculated bymultiplying mass of labelled antigen bound by serum dilution.

A radioimmunoassay system employing I¹²⁵ HCG and antisera raised topeptide conjugates was used to determine the relative ability of HCG andpeptides to compete with labeled HCG. Peak antibody levels from eachrabbit were evaluated in these studies. Antigens and antisera containedin phosphate-buffered saline (pH 7.4) BSA (1%) were added to test tubesand incubated at 4° for 5 days. Separation of free and bound tracer HCGwas accomplished by the addition of sheep anti-rabbit gamma globulinserum and further incubated for 48 hours followed by centrifugation.Assessment of parallelism of dose response curves was accomplished usingmethods described in Rodbard, D. in: Odell, W. D. and Daughaday, W. H.,eds., "Competitive Protein Binding Assays," J. B. Lippincott, Phila. Pa.(1971). The ability of unlabelled HCG and peptides to compete with I¹²⁵HCG for antibody binding sites was expressed as moles of unlabeledantigen, per mole of unlabeled HCG, required to reduce the binding oflabeled HCG by 50%. For this purpose molecular weights for HCG, P-1,P-2, and P-3 of 38,000, 7,000, 3,990, and 3,990 respectively were used.The molecular weight of the P-1 peptide was an estimate since thecontribution of the 4 carbohydrate chains to its size was notdetermined. Four radioimmunoassays were performed with each of the 11antisera studied and the results presented as the mean of the fourvalues.

RESULTS

Parallel dose response curves of HCG and peptides were observed in allradioimmunoassays. In the assay system employed, 200-400 moles ofunlabeled HCG was required per mole of labeled HCG at 50% binding of thelatter to antisera. There was no detectable difference among antisera tothe 3 peptide conjugates in the ability of intact HCG to compete withlabeled hormone for antibody binding sites.

Data obtained from comparing the ability of HCG and peptides to competewith I¹²⁵ HCG for binding to anti-peptide sera revealed some qualitativedifferences in the antisera (Table 2). Much larger quantities of P-2peptide and P-3 peptide were required to reduce I¹²⁵ HCG binding thanwas required by P-1 peptide when sera against the P-1 peptide wastested. While similar quantities of P-2 and P-3 peptides were requiredto inhibit one mole of labeled HCG binding, these were 2-10 times theamounts required by the P-1 peptide.

Differences in the quantities of peptides required to compete with anequivalent mass of labeled HCG were less using antisera raised tocarbohydrate-free natural peptide (P-2). More P-1 peptide was needed foran equal reduction in binding than the other 2 peptides. No significantdifference could be detected in the quantities of P-2 or P-3 peptidesrequired among the 3 antisera tested.

Approximately 1.5-2.0 times as much P-1 peptide was required to competeequally with I¹²⁵ HCG for antibodies raised to the P-3 peptide but P-2peptide reacted nearly as well as did the synthetic peptide.

                  Table 3                                                         ______________________________________                                        Mean Quantities of HCG and 109-145                                            C-terminal β-HCG Peptides                                                Required to Compete with I.sup.125 HCG                                        at 50% Binding of Labelled Hormone                                            Unlabelled Antigens                                                           Antisera                                                                             HCG        P-1       P-2     P-3                                              mol/mol    mol/mol   mol/mol mol/mol                                   Rabbit HCG I.sup.125                                                                            HCG I.sup.125                                                                           HCG I.sup.125                                                                         HCG I.sup.125                             No.    (X ± SE)                                                                              (X ± SE)                                                                             (X ± SE)                                                                           (X ± SE)                               ______________________________________                                        Anti P-1                                                                       78    284 (12.6) 430 (11.8)                                                                              4565 (200.8)                                                                          3628 (154.1)                               79    350 (13.5) 404 (18.5)                                                                              855 (33.4)                                                                            881 (42.2)                                171    403 (17.7) 343 (9.9) 899 (35.1)                                                                            759 (37.1)                                173    377 (16.5) 320 (13.9)                                                                              1448 (72.4)                                                                           1536 (73.7)                               Anti P-2                                                                       93    247 (11.8) 385 (18.2)                                                                              264 (12.5)                                                                            268 (12.73)                                94    294 (14.1) 431 (15.5)                                                                              362 (15.2)                                                                            329 (13.8)                                252    201 (9.6)  296 (12.4)                                                                              216 (7.7)                                                                             205 (9.0)                                 Anti P-3                                                                      405    496 (23.6) 998 (47.4)                                                                              628 (27.6)                                                                            309 (13.6)                                411    489 (20.5) 1200 (50.4)                                                                             678 (29.7)                                                                            413 (16.1)                                416    364 (13.1) 581 (20.9)                                                                              400 (14.4)                                                                            271 (12.8)                                417    340 (14.9) 474 (18.4)                                                                              176 (6.8)                                                                             105 (4.6)                                 ______________________________________                                    

DISCUSSION

Despite low levels of antibodies obtained in this study, thecarbohydrate-containing peptide was not more immunogenic than thosewithout this moiety when conjugates to both were prepared in the samemanner.

From these studies, it can be concluded that although antibodies tocarbohydrate free peptides are qualitatively different than those to thenatural peptide, antisera generated to the synthetic peptide reactedwith HCG as well as antisera to natural peptides and equivalent tonatural and synthetic peptides elicited similar anti-HCG levels inrabbits.

EXAMPLE XXVII

In this Example, a polypeptide fragment structure having an --SH groupis activated utilizing the following reagent: ##STR8## A solution of thereagent (1.2 eq. per --SH group in the polypeptide) in a suitable watermiscible organic solvent, such as dioxane, is added to a solution of thepolypeptide fragment structure, e.g. Structure (XII) (which has had itsamino groups blocked) in aqueous buffer at pH 6.5. After 2 hours, thesolvent is removed at a temperature of less than 30° C. under vacuum,and to the residue are added water and ethyl ether (1:1). The aqueouslayer is separated and its pH adjusted to approximately 8.5 by theaddition of sodium hydroxide solution and this alkaline mixture is addedrapidly to an aqueous solution of the carrier, e.g. the above describedinfluenza subunit, maintained at pH 8.5 by a suitable buffer. After afurther 4 hours, the conjugate is isolated, by gel filtration.

EXAMPLE XXVIII

With the following reagent: ##STR9## a solution or suspension of acarrier containing no sulfhydryl groups such as Flagellin in a suitableaqueous buffer at a pH 6.5 is treated with the required (1.2 eq/--NH₂desired to be reacted) amount of a solution of the reagent indimethylformamide. After 1 hour, the modified carrier is isolated bycolumn chromatography and added to buffer at pH 6-7. This is thentreated with a solution of the selected fragment (containing sulfhydrylgroups) in the same buffer and the reaction is allowed to proceed for 12hours before the conjugate is isolated by column chromatography.

EXAMPLE XXIX

Modification of non-sulfhydryl containing peptide fragments [e.g.structure (II)] or a carrier such as Flagellin to a sulfhydrylcontaining one via "thiolactonization" is carried out as follows.

The peptide is dissolved in a 1 M aqueous solution of imidazolecontaining 0.5% of ethylenediamine tetraacetic acid at a pH of 9.3 underan atmosphere of nitrogen and a 100 fold excess of N-acetylhomocysteinethiolactone is added in three portions at eight hour intervals. After atotal of 30 hours, the pH is adjusted to 3-4 with acetic acid and themodified peptide is isolated by gel chromatography and elution with 0.5M acetic acid.

EXAMPLE XXX

The carrier protein is reacted with the N-hydroxysuccinimide ester of ahalo-(either chloro, bromo or iodo) acetic acid in the general proceduredescribed in the first part of Example XXVIII thus yielding a modifiedcarrier containing the required number of halomethyl alkylating groupsas desired.

To a solution of the sulfhydryl containing peptide [e.g. structure(XII)] in a phosphate buffer at pH 6.5-7.0 under nitrogen at roomtemperature is added an aqueous solution or suspension of the modifiedcarrier prepared above. The mixture is stirred for 12 hours. It is thenwashed with ethyl acetate and the conjugate contained in the aqueousphase is purified by dialysis, gel chromatography and lyophilization.

Should neither the carrier nor polypeptide fragment contain a sulfhydrylgroup, one may be introduced into either of them by the standardprocedures such as "thiolactonization" described above under ExampleXXIX.

What is claimed is:
 1. A method for controlling fertility in primateanimals having naturally occurring endogenous Chroionic Gonadotropinhormone by neutralizing the biological action of the endogenous hormone,comprising the steps of:administering to said primate animal animmunologically effective amount of the hormone, a subunit thereof, apeptide fragment of the subunit or a synthetically derived peptidehaving a sequence analogous to at least a portion of said subunit; saidhormone, subunit, fragment or synthetically derived peptide beingmodified by the coupling thereof with a non-endogenous material toeffect the formation, following said administration, of antibodieshaving a specificity to endogenous Chorionic Gonadotropin; therebyinhibiting the fertility of said primate animal by preventing one ormore normal biological functions of the endogenous ChorionicGonadotropin hormone.
 2. The method of claim 1 wherein said modifiedhormone, subunit, fragment or synthetically derived peptide isadministered in combination with an adjuvant.
 3. The method ofcontrolling fertility in primate animals having naturally occuringendogenous Chorionic Gonadotropin hormone comprising the stepsof:providing a quantity of the hormone, a subunit thereof, a peptidefragment thereof, or a synthetically derived peptide having a sequenceanalogous to at least a portion of said subunit and being non-antigenicwithin said primate animals; modifying said hormone, subunit, fragment,or synthetically derived peptide by the coupling thereof with anon-endogenous material; administering to said primate animal animmunologically effective amount of said modified hormone, subunit,fragment, or synthetically derived peptide; thereby inhibiting thefertility of said primate animals by preventing one or more normalbiological functions of endogenous Chorionic Gonadotropin.
 4. The methodof claim 3 wherein said modified hormone, subunit, fragment orsynthetically derived peptide is administered in combination with anadjuvant.
 5. The method of claim 3 wherein said non-endogenous materialis coupled with said hormone, subunit, fragment or synthetically derivedpeptide through a sulfhydryl-group linkage.
 6. The method of claim 3wherein said hormone, subunit, fragment or synthetically derived peptideis one having a sulfhydryl group therewithin, and said coupling iscarried out by the treatment thereof with an activator of the structure##STR10## wherein X represents a non-reacting connective entity so as toeffect reaction of the maleiimide group of the activator with a saidsulfhydryl group; andtreating the resultant activated hormone, subunit,fragment or synthetically derived peptide with said non-endogenousmaterial.
 7. The method of claim 6 wherein said non-reacting connectiveentity, X, comprises an amino acid chain.
 8. The method of claim 7wherein said amino acid chain is a Prolene spacer chain.
 9. The methodof claim 3 wherein said non-endogenous material comprises sucrosecopolymerized with epichlorohydrin.
 10. The method of claim 3 whereinsaid non-endogenous material comprises (poly)tyrosine, (poly)alanine,(poly)dextran, thyroglobulin, or a combination thereof.
 11. The methodof claim 6 wherein said non-endogenous material comprises the protein,flagellin.
 12. The method of claim 6 wherein said non-endogenousmaterial material comprises an influenza virus.
 13. The method of claim12 in which said influenza virus is an influenza subunit incorporatingsubstantially only the viral proteins Haemagglutin and Neuraminease. 14.The method of claim 6 wherein said non-endogenous material is diptheriatoxiod.
 15. The method of claim 6 wherein said non-endogenous materialis a cholera organism.
 16. The method of claim 6 wherein said activatedhormone, subunit, fragment or synthetically derived peptide is treatedwith said non-endogenous material at slightly alkaline pH.
 17. Themethod of claim 3 or 6 wherein said fragment which is modified is of thechemicalconfiguration:Thr-Cys-Asp-Asp-Pro-Arg-Phe-Gln-Asp-Ser-Ser-Ser-Ser-Lys-Ala-Pro-Pro-Pro-Ser-Leu-Pro-Ser-Pro-Ser-Arg-Leu-Pro-Gly-Pro-Ser-Asp-Thr-Pro-Ile-Leu-Pro-Gln;Asp-Asp-Pro-Arg-Phe-Gln-Asp-Ser-Pro-Pro-Pro-Cys-Pro-Pro-Pro-Ser-Asp-Thr-Pro-Ile-Leu-Pro-Gln;Asp-Asp-Pro-Arg-Phe-Gln-Asp-Ser-Pro-Pro-Pro-Pro-Pro-Pro-Cys-Pro-Pro-Pro-Pro-Pro-Pro-Ser-Asp-Thr-Pro-Ile-Leu-Pro-Gln;Asp-His-Pro-Leu-Thr-Aba-Asp-Asp-Pro-Arg-Phe-Gln-Asp-Ser-Ser-Ser-Ser-Lys-Als-Pro-Pro-Pro-Ser-Leu-Pro-Ser-Pro-Ser-Arg-Leu-Pro-Gly-Pro-Ser-Asp-Thr-Pro-Ile-Leu-Pro-Gln-Pro-Pro-Pro-Pro-Pro-Pro-Cys;Asp-His-Pro-Leu-Thr-Aba-Asp-Asp-Pro-Arg-Phe-Gln-Asp-Ser-Ser-Ser-Ser-Lys-Ala-Pro-Pro-Pro-Ser-Leu-Pro-Ser-Pro-Ser-Arg-Leu-Pro-Gly-Pro-Ser-Asp-Thr-Pro-Ile-Leu-Pro-Gln-Cys;orCys-Pro-Pro-Pro-Pro-Pro-Pro-Asp-Asp-Pro-Arg-Phe-Gln-Asp-Ser-Ser-Ser-Ser-Lys-Ala-Pro-Pro-Pro-Ser-Leu-Pro-Ser-Pro-Ser-Arg-Leu-Pro-Gly-Pro-Ser-Asp-Thr-Pro-Ile-Leu-Pro-Gln.18. The method of claim 3 wherein said fragment which is modified is ofthe chemicalconfiguration:Asp-Asp-Pro-Arg-Phe-Gln-Asp-Ser-Ser-Ser-Ser-Lys-Ala-Pro-Pro-Pro-Ser-Leu-Pro-Ser-Pro-Ser-Arg-Leu-Pro-Gly-Pro-Ser-Asp-Thr-Pro-Ile-Leu-Pro-Gln;Gln-Asp-Ser-Ser-Ser-Ser-Lys-Ala-Pro-Pro-Pro-Ser-Leu-Pro-Ser-Pro-Ser-Arg-Leu-Pro-Gly-Pro-Ser-Asp-Thr-Pro-Ile-Leu-Pro-Gln,Phe-Gln-Asp-Ser-Ser-Ser-Ser-Lys-Ala-Pro-Pro-Pro-Ser-Leu-Pro-Ser-Pro-Ser-Arg-Leu-Pro-Gly-Pro-Ser-Asp-Thr-Pro-Ile-Leu-Pro-Gln;orAsp-Asp-Pro-Arg-Phe-Gln-Asp-Ser-Ser-Ser-Ser-Lys-Als-Pro-Pro-Pro-Ser-Leu-Pro-Ser.19. The method of claim 3 wherein said non-endogenous material is onehaving an amino group and said coupling is carried out by activatingsaid non-endogenous material with an activator of the structure:##STR11## where X represents a non-reacting connective entity so as toeffect reaction of the activator with said amino group; andtreating theactivated non-endogenous material with said hormone, subunit, fragmentor synthetically derived peptide which has a sulfhydryl group.
 20. Themethod of claim 19 wherein said activation is carried out under neutralor acid conditions.
 21. The method of claim 3 wherein said modificationis carried out to effect the constitution of two or more immunologicaldeterminants effective to elicit antibody response to the endogenoushormone, Chorionic Gonadotropin.
 22. The method of claim 3 wherein:saidfragment or synthetically derived peptide which is modified is of thechemicalconfiguration:Cys-Pro-Pro-Pro-Pro-Pro-Pro-Ser-Asp-Thr-Pro-Ile-Leu-Pro-Glnand said administration thereof is in combination with another,different modified Chorionic Gonadotropin hormine, subunit thereof,peptide fragment of the said subunit, or a synthetically derived peptidehaving a sequence at least analogous to at least a portion of thefragment, the said modification of which effects the constitution of atleast one immunological determinant.
 23. The method of claim 3wherein:said fragment or synthetically derived peptide which is modifiedis of the chemicalconfiguration:Asp-Asp-Pro-Arg-Phe-Gln-Asp-Ser-Pro-Pro-Pro-Pro-Pro-Pro-Cysand said administration thereof is in combination with another,different modified Chorionic Gonadotropin hormine, subunit thereof,peptide fragment of the said subunit, or a synthetically derived peptidehaving a sequence at least analogous to at least a portion of thefragment, the said modification of which effects the constitution of atleast one immunological determinant.
 24. The method of claim 1 whereinsaid animal is human and said hormone is human Chorionic Gonadoptropin.